Biochemistry Flashcards
What are the four groups attached to the central (alpha) carbon of a proteinogenic amino acid?
Amino group (NH2), carboxylic acid group (COOH), hydrogen and R group
Are eukaryotic amino acids L or D?
L
Are eukaryotic amino acids R or S?
S
What is the only eukaryotic amino acid that is R?
Cysteine
What is the only achiral amino acid?
Glycine
Nonpolar, non-aromatic amino acids
Glycine, alanine, valine, leucine, isoleucine, methionine, proline
Nonpolar, aromatic amino acids
Tryptophan and phenylalanine
Polar, aromatic amino acids
Tyrosine
Polar, non-aromatic amino acids
Serine, threonine, asparagine, glutamine and cysteine
Negatively charged (acidic) amino acids
Aspartate and glutamate
Positively charged (basic) amino acids
Lysine, arginine and histidine
What kind of amino acid is glycine?
Nonpolar, non-aromatic
What kind of amino acid is alanine?
Nonpolar, non-aromatic
What kind of amino acid is valine?
Nonpolar, non-aromatic
What kind of amino acid is leucine?
Nonpolar, non-aromatic
What kind of amino acid is isoleucine?
Nonpolar, non-aromatic
What kind of amino acid is methionine?
Nonpolar, non-aromatic
What kind of amino acid is proline?
Nonpolar, non-aromatic
What kind of amino acid is tryptophan?
Nonpolar, aromatic
What kind of amino acid is phenylalanine?
Nonpolar, aromatic
What kind of amino acid is tyrosine?
Polar, aromatic
What kind of amino acid is serine?
Polar, non-aromatic
What kind of amino acid is threonine?
Polar, non-aromatic
What kind of amino acid is asparagine?
Polar, non-aromatic
What kind of amino acid is glutamine?
Polar, non-aromatic
What kind of amino acid is cysteine?
Polar, non-aromatic
What kind of amino acid is aspartate?
Negatively charged (acidic)
What kind of amino acid is glutamate?
Negatively charged (acidic)
What kind of amino acid is lysine?
Positively charged (basic)
What kind of amino acid is arginine?
Positively charged (basic)
What kind of amino acid is histidine?
Positively charged (basic)
Glycine R group
H
Alanine R group
CH3
Valine R group
CH - (CH3)2
Leucine R group
CH2 - CH - (CH3)2
Isoleucine R group
CH - [CH3, CH2 - CH3]
Methionine R group
CH2 - CH2 - S - CH3
Proline R group
Cyclic
Tryptophan R group
CH2 - double ring system with N
Phenylalanine R group
CH2 - benzene ring
Tyrosine R group
CH2 - Phenyl
Serine R group
CH2 - OH
Threonine R group
C - [H, OH, CH3]
Asparagine R group
CH2 - C - [NH2, O]
Glutamine R group
CH2 - CH2 - C - [NH2, O]
Cysteine R group
CH2 - SH
Aspartic acid R group
CH2 - COO-
Glutamic acid R group
CH2 - CH2 - COO-
Lysine R group
(CH2)4 - NH3+
Arginine R group
(CH2)3 - NH - C - [NH, NH3+]
Histidine R group
CH2 - imidazole ring
Alanine
Ala, A, non polar, non-aromatic
Arginine
Arg, R, basic
Asparagine
Asn, N, polar, non-aromatic
Aspartate
Asp, D, acidic
Cysteine
Cys, C, polar, non-aromatic
Glutamate
Glu, E, acidic
Glutamine
Gln, Q, polar, non-aromatic
Glycine
Gly, G, non polar, non-aromatic
Histidine
His, H, basic
Isoleucine
Ile, I, nonpolar, non-aromatic
Leucine
Leu, L, nonpolar, non-aromatic
Lysine
Lys, K, basic
Methionine
Met, M, nonpolar, non-aromatic
Phenylalanine
Phe, F, nonpolar, aromatic
Proline
Pro, P, nonpolar, non-aromatic
Serine
Ser, S, polar, non-aromatic
Threonine
Thr, T, polar, non-aromatic
Tryptophan
Trp, W, nonpolar, aromatic
Tyrosine
Tyr, Y, polar, aromatic
Valine
Val, V, nonpolar, non-aromatic
Amino acid
Makes up peptide chains (i.e. proteins)
Dipeptide
Two amino acids joined by a peptide bond
Tripeptide
Three amino acids joined by peptide bonds
Oligopeptide
Less than 20 amino acids joined by peptide bonds
Polypeptide
More than 20 amino acids joined by peptide bonds
What molecule is released during the formation of a peptide bond?
Water
Primary protein structure
Linear amino acid sequence
Secondary protein structure
Local structure determined by nearby amino acids
Subtypes of secondary protein structure
Alpha-helices and beta-pleated sheets
Stabilizing bonds of the primary protein structure
Peptide (amide) bonds
Stabilizing bonds of the secondary protein structure
Hydrogen bonds between amino groups and nonadjacent carboxyl groups
What role does proline serve in the secondary protein structure?
Its rigid structure causes it to introduce kinks in alpha-helices, or create turns in beta-pleated sheets
Tertiary protein structure
Protein folding (three-dimensional shape of protein)
Quaternary protein structure
Interaction between separate subunits of a multisubunit protein
Subtypes of tertiary protein structure
Hydrophobic interactions, acid-base/salt bridges, disulfide links
Stabilizing bonds of the tertiary protein structure
Hydrogen bonds, van der Waals forces, ionic bonds and covalent bonds
Stabilizing bonds of the quaternary protein structure
Hydrogen bonds, van der Waals forces, ionic bonds and covalent bonds
Why are proteins denatured by heat?
Heat increases the average kinetic energy, thereby distrusting hydrophobic interactions
Why are proteins denatured by solutes?
Solutes disrupt elements of secondary, tertiary and quaternary structures
Isoelectric point (pI) of neutral amino acid
= (pKa, NH3+ group + pKa, COOH group) / 2
Isoelectric point (pI) of acidic amino acid
= (pKa, R group + pKa, COOH group) / 2
Isoelectric point (pI) of basic amino acid
= (pKa, R group + pKa, NH3+ group) / 2
What does the R group on an amino acid do?
Determine the chemistry and function of that amino acid
Are prokaryotic amino acids D or L?
D
What happens to an amino acid in low (acidic) pH?
It is fully protonated
What happens to an amino acid in pH closer to the amino acid’s pI?
The amino acid is neutral (zwitterion)
What happens to an amino acid in high (basic) pH?
It is fully deprotonated
What does the titration curve of an amino acid look like?
Flat at pKa values and vertical in pI values
How to form a peptide bond?
Through a condensation or dehydration reaction, releasing one molecule of water
The nucleophile (amino group) of one amino acid attacks the electrophile (carboxyl group) of another amino acid
What is the nucleophilic group on an amino acid?
Amino group
What is the electrophilic group on an amino acid?
Carbonyl carbon of the carboxyl group
Why are amide bonds rigid?
Because of resonance
How to break a peptide bond?
Hydrolysis reaction
Alpha-helices
Clockwise coils around a central axis
Beta-pleated sheets
Rippled strands that can be parallel or antiparallel
Can proline interrupt secondary protein structure?
Yes because of its rigid structure
Hydrophobic interactions
Push hydrophobic R groups to the interior of a protein, which increases entropy of the surrounding water molecules and creates negative Gibbs free energy
What do hydrophobic interactions result in?
Increase in entropy of the surrounding water and negative Gibbs free energy
When do disulfide bonds occur?
When two cysteine molecules are oxidized and create a covalent bond to form cystine
Conjugated proteins
Proteins with covalently attached molecules
Prosthetic group
Tightly bound cofactors or coenzymes that are necessary for enzyme function
The molecule attached to a conjugated protein
Can be metal ion, vitamin, lipid, carbohydrate or nucleic acid
Denaturation
Loss of three-dimensional protein structure
Which amino acids have chiral carbons in their side chain?
Threonine and isoleucine
What is the reason behind protein conjugation?
Directing protein to a particular organelle, directing protein to the cell membrane and adding a cofactor needed to protein activity
How do enzymes function as biological catalysts?
Improve the environment in which a particular reaction takes place, which lowers its activation energy
They are regenerated at the end of the reaction to their original form
They can form transient covalent bonds with substrates
They can act as electron donors or receptors to allow the reaction to proceed
Enzyme specificity
Enzymes are specific for certain kinds of compounds and perform certain kinds of reactions
The six classes of enzymes
Ligase, isomerase, lyase, hydrolase, oxidoreductase and transferase
Ligase
Addition or synthesis reactions, generally between large molecules (often of the same type); often require ATP
Isomerase
Rearrangement of bonds within a compound
Catalyze the interconversion of isomers, including both constitutional isomers and stereoisomers
Lyase
Cleavage of a single molecule into two products, or synthesis of small organic molecules
Does not use water or transfer electrons
The opposite of synthesis
Often form cyclic compounds or double bonds in products
Hydrolase
Breaking of a compound into two molecules using the addition of water
Oxidoreductase
Oxidation-reduction reactions (transferring electrons)
Transferase
Movement of a functional group from one molecule to another
In what ways to enzymes affect the thermodynamics vs. the kinetics of a reaction?
Enzymes only affect the kinetics of a reaction, by lowering the activation energy (i.e. lowering the transition state energy). Enzymes do not affect the thermodynamics of a reaction since they do not change delta G, delta H, the equilibrium constant, the equilibrium position or the concentrations of reactants and products.
Catalysts
Reduce the activation energy of a reaction, thus speeding up the reaction
They are not used up in the course of the reaction
Lock and key model
Active site of enzyme fits exactly around substrate
No alterations to tertiary or quaternary structure of enzyme
Less accurate model
Induced fit model
Active site of enzyme molds itself around substrate only when substrate is present
Tertiary and quaternary structure is modified for enzyme function
More accurate model
Cofactors
Inorganic (minerals) activators of enzymes
Induce conformational change in the enzyme to promote its activity
Coenzymes
Organic (vitamins) activators of enzymes
Induce conformational change in the enzyme to promote its activity
What are the effects of increasing substrate concentration on enzyme kinetics?
When the substrate concentration is low, an increase in substrate concentration causes a proportional increase in enzyme activity
When the substrate concentration is high and the enzyme is saturated, increasing substrate concentration will have no effect on enzyme activity since v max has already been attained
What are the effects of increasing enzyme concentration on enzyme kinetics?
Increasing enzyme concentration will always increase v max, regardless of the starting concentration of enzyme
Michaelis-Menten plot
v vs [S]
Hyperbolic curve for monoatomic enzymes
Lineweaver-Burk plot
1/v vs 1/[S]
Linear plot
Km
A measure of an enzyme’s affinity for its substrate
The substrate concentration at which an enzyme is functioning at half of its maximal velocity
What does an increase Km signify?
Decrease in an enzyme’s affinity for its substrate
What is the x-intercept of a Lineweaver-Burk plot?
- 1/Km
What is the y-intercept of a Lineweaver-Burk plot?
1/v max
Enzyme cooperativity
The interactions between subunits in a multi-subunit enzyme or protein
The binding of a substrate to one subunit induces a change in the other subunits from the T(tense) state to the R (relaxed) state, which encourages binding of substrate to the other subunits
The unbinding of a substrate from one subunit indices a change from R to T in the remaining subunits, promoting unbinding of substrate from the remaining subunits
Temperature’s effect on enzyme function
As temperature increases, enzyme activity generally increases (doubling approximately every 10 C)
Above body temperature, however, enzyme activity quickly drops off as the enzyme denatures
pH’s effect on enzyme function
Enzymes are maximally active within a small pH range
Outside of the range, enzyme activity drops quickly with changes in pH as the ionization of the active site changes and the protein is denatured
Salinity
Changes in salinity can disrupt bonds within an enzyme, causing disruption of tertiary and quaternary structure, which leads to loss of enzyme function
Ideal temperature for enzyme function
98.6 F = 37 C = 310 K
Ideal pH for most enzymes
7.4
Ideal pH for gastric enzymes
2
Ideal pH for pancreatic enzymes
8.5
Feedback inhibition
The product of an enzymatic pathway turning off enzymes further back in that same pathway
Helps maintain homeostasis: as product levels rise, the pathway creating that product is appropriately down-regulated
Types of reversible inhibitors
Competitive, noncompetitive, mixed and uncompetitive
Irreversible inhibition
The prolonged or permanent inactivation of an enzyme, such that is cannot be easily renatured to gain function
New enzyme molecules must be synthesized for the reaction to occur again
Transient enzyme modifications
Allosteric activation
Allosteric inhibition
Covalent enzyme modifications
Phosphorylation
Glycosylation
Why are some enzymes released as zymogens?
It is critical that certain enzymes (like the digestive enzymes of the pancreas) remain inactive until arriving at their target site
Is allosteric activation a transient or covalent enzyme modification?
Transient
Is allosteric inhibition a transient or covalent enzyme modification?
Transient
Is phosphorylation a transient or covalent enzyme modification?
Covalent
Is glycosylation a transient or covalent enzyme modification?
Covalent
Zymogens
Precursors of an active enzyme
They are secreted in an inactive form and are activated by cleavage
Michaelis-Menten rates
E + S ES –(K3)–> E + P
Michaelis-Menten equation
v = ( v max [S]) / (Km + [S])
Synthesis
The opposite of lyases
Exergonic reaction
Release energy
delta G is negative
Delta G is negative
Exergonic reaction
Do enzymes stabilize the transition state?
Yes
They provide a favorable micro-environment or bonding with substrate molecules
Active site
The site of catalysis in an enzyme
Saturation kinetics
As substrate concentration increases, the reaction rate does as well until a maximum value is reached
What kind of Michaelis-Menten graph do cooperative enzymes make?
Sigmoidal because of the change in activity with substate binding
Does temperature affect enzyme activity in vivo or in vitro?
In vivo
Does pH affect enzyme activity in vivo or in vitro?
In vivo
Does salinity affect enzyme activity in vivo or in vitro?
In vitro
In vitro
In the lab
In vivo
In the body
Reversible inhibition
The ability to replace the inhibitor with a compound greater affinity or to remove it using mild laboratory treatment
Competitive inhibition
Results when the inhibitor is similar to the substrate and binds at the active site
How can competitive inhibition be overcome?
Adding more substrate
What happens to v max in competitive inhibition?
Nothing
What happens to Km in competitive inhibition?
Increases
Noncompetitive inhibition
Results when the inhibitor binds with equal affinity to the enzyme (at an allosteric site) and the enzyme-substrate complex
What happens to v max in noncompetitive inhibition?
Decreases
What happens to Km in noncompetitive inhibition?
Nothing
Mixed inhibition
Results when the inhibitor binds with unequal affinity to the enzyme (at an allosteric site) and the enzyme-substrate complex
What happens to v max in mixed inhibition?
Decreases
What happens to Km in mixed inhibition?
Increases if the inhibitor prefers to bind to the enzyme at an allosteric site
Decreases if the inhibitor prefers to bind to the enzyme-substrate complex
Uncompetitive inhibition
Results when the inhibitor binds only with the enzyme-substrate complex
What happens to v max in uncompetitive inhibition?
Decreases
What happens to Km in uncompetitive inhibition?
Decreases
Allosteric activation
Activators bind to the allosteric site of an enzyme, increasing enzyme affinity to substrate
Allosteric inhibition
Inhibitors bind to the allosteric site of an enzyme, decreasing enzyme affinity to substrate
Phosphorylation
Covalent modification of an enzyme with phosphate
Can alter the activity or selectivity of an enzyme
Glycosylation
Covalent modification of an enzyme with carbohydrate
Can alter the activity or selectivity of an enzyme
Apoenzyme
An enzyme devoid of its necessary cofactor and is catalytically inactive
What determines enzyme specificity?
The three-dimensional shape of the active site
Cytoskeletal proteins
Fibrous
Have repeating domains
Function in cellular motility
Motor proteins
Have one or more heads capable of force generation through a conformational change
Have catalytic activity, acting as ATPases to power movement
Are motor proteins enzymes?
No
What could permit a binding protein involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound?
If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrate will be bound despite a low affinity
Cell adhesion molecules (CAM)
Allow cells to bind to other cells or surfaces
Cadherin, integrin and selectin
What type of cell adhesion does cadherin form?
Two cells of the same or similar type using calcium
What type of cell adhesion does integrin form?
One cell to proteins in the extracellular matrix
What type of cell adhesion does selectin form?
One cell to carbohydrates, usually on the surface of other cells
When an antibody binds to its antigen, what are the three possible outcomes of this interaction?
Antigen-antibody interactions can result in neutralization of the pathogen or toxin, opsonization (marking) of the antigen for destruction, or creation of insoluble antigen-antibody complexes that can be phagocytize and digested by macrophages (agglutination).
Enzyme
A protein or RNA molecule with catalytic activity
Do motor proteins have catalytic activity?
No
Enzyme-linked receptors
Participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades Autoactivity Enzymatic activity Extracellular domain Transmembrane domain Ligand binding
G protein-coupled receptors
Have membrane-bound protein associated with a trimetric G protein Initiate second messenger systems Two-protein complex Dissociation upon activation Extracellular domain Transmembrane domain Ligand binding
What type of ion channel is active at all times?
Ungated channels
How do transport kinetics differ from enzyme kinetics?
Transport kinetics display both Km and v max values. They also can be cooperative, like some binding proteins. However, transporters do not have analogous Keq values for reactions because there is no catalysis.
What separation methods can be used to isolate a protein on the bases of isoelectric point?
Isoelectric focusing and ion-exchange chromatography can separate proteins based on charge
What are the relative benefits of native PAGE compared to SDS-PAGE?
Native PAGE allows a complete protein to be recovered after analysis; it also more accurately determines the relative globular size of proteins. SDS-PAGE can be used to eliminate conflation from mass-to-charge ratios.
What are two potential drawbacks of affinity chromatography?
The protein of interest may not elute from the column because its affinity is too high
The protein may be permanently bound to the free receptor in the eluent
In size-exclusion chromatography, do the largest or the smallest molecules elute first?
The largest molecules elute first since the size-exclusion chromatography trap smaller particles, retaining them in the column
How does isoelectric focusing separate proteins?
Separate proteins by charge
The protein migrates towards an electrode until pH = pI of the protein
How does ion-exchange chromatography separate proteins?
Separate proteins by charge
How is the charge of a protein determined?
By the protein’s isoelectric point (pI)
Why are proteins analyzed after isolation?
Protein isolation is generally only the first step in an analysis. The protein identity must be confirmed by amino acid analysis or activity. With unknown proteins, classification of their features is generally desired.
What factors would cause an activity assay to display lower activity than expected after concentration determination?
Contamination of the sample with detergent or SDS could yield an artificially increased protein level, leading to lower activity than expected (because the protein concentration was calculated as higher than its actual value). Alternatively, the enzyme could have been denatured during isolation and analysis.
Does the Edman degradation proceed from the carboxy (C-) terminus or the amino (N-) terminus?
Amino (N-) terminus
Migration velocity (v)
= Ez / f
Structural proteins
Compose the cytoskeleton, anchoring proteins and must of the extracellular matrix
Fibrous
Common structural proteins
Collagen, elastin, keratin, actin and tubulin
Applications of motor proteins
Muscle contractions, vesicle movement within cells, cell motility
Common motor proteins
Myosin, kinesin and dynein
Binding proteins
Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state
Cadherins
Calcium-dependent glycoproteins that hold similar cells together
Integrins
Have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix. Some also have signaling capabilities
Selectins
Allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system
Antibodies (immunoglobulins, Ig)
Used by the immune system to target a specific antigen, which may be a protein on the surface of a pathogen (invading organism) or a toxin
Structure of antibodies (immunoglobulins, Ig)
Contain a constant region and variable region; the variable region is responsible for antigen binding
Two identical heavy chains and two identical light chains form a single antibody; they are held together by disulfide linkages and non covalent interactions
How are the light and heavy chains of an antibody held together?
By disulfide linkages and non-covalent interactions
Ion channels
Used to regulate ion flow into or out of the cells
Types of ion channels
Ungated, voltage-gated and ligand-gated
Ungated channels
Are always open
Voltage-gated channels
Open within a range of membrane potentials
Ligand-gated channels
Open in the presence of a specific binding substance, usually a hormone or neurotransmitter
How does a G-protein- coupled receptor work?
Ligand binding engages the G protein –> GDP is replaced with GTP –> The alpha subunit dissociates from the beta and gamma subunits –> The activated alpha subunit alters the activity of adenylate cyclase or phospholipase C –> GTP is dephosphorylated to GDP –> The alpha subunit rebinds to the beta and gamma subunits
Electrophoresis
Uses a Gell matrix to observe the migration of proteins in response to an electric field
Native PAGE
Maintains the protein’s shape, but results are difficult to compare because the mass-to-charge ration differs for each protein
SDS-PAGE
Denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel
Chromatography
Separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase
Column chromatography
Uses beads of a polar compound, like silica or alumina (stationary phase), which a non polar solvent (mobile phase)
Ion-exchange chromatography
Uses a charged column and a variably saline eluent
Size-exclusion chromatography
Relies on porous beads
Larger molecules elute first because they are not trapped in the small pores
Affinity chromatography
Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest
How is protein structure determined?
X-ray crystallography (after the protein has been isolated)
NMR can also be used
How is amino acid composition determined?
Simple hydrolysis
How is amino acid sequence determined?
Sequential degradation
Example of methods of protein degradation
Edman degradation
How is enzyme activity determined?
Following the process of a known reaction and color change
How is protein concentration determined?
Colorimetrically (either by UV spectroscopy or color change in a reaction)
Bradford protein assay test
BCA assay
Lowry reagent assay
Bradford protein assay
Uses color change from brown-green to blue
BCA Assay
The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid.
Lowry reagent assay
The Lowry protein assay uses copper, which bonds with the peptide bonds in proteins under alkaline conditions. This forms a monovalent copper ion which can then react with the Folin reagent, which in turn can be reduced into a blue colored substance. This blue color can be measured using a spectrophotometer to determine the concentration of blue in the sample. Thus, the concentration of protein can be determined.
What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE?
It stabilizes proteins to give them uniformly negative charges, so the separation is based purely on size
What are the most prevalent extracellular proteins?
Keratin, elastin and collagen
What are the primary cytoskeletal proteins?
Tubulin and actin
What kind of protein is myosin?
Motor protein
Do antibodies label antigens for targeting by other immune cells?
Yes
Do antibodies cause agglutination by interaction with antigen?
Yes
Do antibodies have two heavy chains and two light chains?
Yes
Can antibodies bind to more than one distinct antigen?
No
Which ion channels are responsible for maintaining the resting membrane potential?
Ungated channels
Which ion channels are involved in cell signaling and pacemaker potentials?
Ligand-gated and voltage-gated channels
How does the gel for isoelectric focusing differ from the gel for traditional electrophoresis?
Isoelectric focusing uses gel with a pH gradient that encourages a variable charge
Which protein properties allow UV spectroscopy to be used as a method of determining concentrations?
Proteins contain aromatic groups in certain amino acids
What property of protein-digesting enzymes allows for a sequence to be determined without fully degrading the protein?
Selectivity
D-stereoisomers of glucose
D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose and D-talose
Which of the diastereomers of D-glucose are considered to be epimers of glucose?
D-allose, D-mannose and D-galactose
What is the enantiomer of D-glucose?
L-glucose
D-allose structure
RRRR
Is D-allose an epimer of D-glucose?
Yes
D-altrose structure
LRRR
Is D-altrose an epimer of D-glucose?
No
D-glucose structure
RLRR
D-mannose structure
LLRR
Is D-mannose an epimer of D-glucose?
Yes
D-gulose structure
RRLR
Is D-gulose an epimer of D-glucose?
No
D-idose structure
LRLR
Is D-idose an epimer of D-glucose?
No
D-galactose structure
RLLR
Is D-galactose an epimer of D-glucose?
Yes
D-talose structure
LLLR
Is D-talose an epimer of D-glucose?
No
What is the less stable anomer of D-glucose in Haworth projection form?
OH at C1: pointing down OH at C2: pointing down OH at C3: pointing up OH at C4: pointing down CH2OH at C5: pointing up
What is the less stable anomer of D-glucose in chair configuration?
All the hydroxyl groups are in the axial position
What is the difference between esterification and glycoside formation?
Esterification is the reaction by which a hydroxyl group reacts with either a carboxylic acid or a carboxylic acid derivative to form an ester. Glycoside formation refers to the reaction between an alcohol and a hemiacetal (or hemiketal) group on a sugar to yield an alkoxy group.
From a metabolic standpoint, does it make sense for carbohydrates to get oxidized or reduced?
Oxidized; because aerobic metabolism requires reduced forms of electron carriers to facilitate processes such as oxidative phosphorylation. Because carbohydrates are a primary energy source, they are oxidized.
Which of the two forms of starch is more soluble in solution?
Amylopectin is more soluble in solution than amylose because of its branched structure. The highly branched structure of amylopectin decreases intermolecular bonding between polysaccharide polymers and increases interaction with the surrounding solution.
Regarding glycogen and amylopectin, which of these two polymers should experience a higher rate of enzyme activity from enzymes that cleave side branches?
Glycogen has a higher rate of enzymatic branch cleavage because it contains significantly more branching than amylopectin.
Number of stereoisomers with common backbone
= 2^n, where n = the number of chiral carbons
How are carbohydrates organized?
By their number of carbon atoms and functional groups
Trioses
Three-carbon sugars
Tetroses
Four-carbon sugars
Aldoses
Sugars with aldehydes as their most oxidized group
Ketoses
Sugars with ketones as their most oxidized group
What is the nomenclature of sugars based on?
D- and L-forms of glyceraldehydes
D-sugars
Sugars with the highest-numbered chiral carbon with the -OH group on the right in a Fischer projection
L-sugars
Sugars with the highest-numbered chiral carbon with the -OH group on the left in a Fischer projection
Enantiomers of sugars
D- and L-forms
Diastereomers
“Nonsuperimposable configurations of molecules with similar connectivity
Differ in at least one, but not all, chiral carbons”
Subtypes of diastereomers
Epimers and anomers
Epimers
A subtype of diastereomerts that differ at exactly one chiral carbon
Anomers
A subtype of diastereomers that differ at the anomeric carbon
Cyclization
Describes the ring formation of carbohydrates from their straight-chain forms
Anomeric carbon
The new chiral center formed in ring closure; it was the carbon containing the carbonyl in the straight-chain form
Alpha-anomers
Have the -OH on the anomeric carbon trans to the free -CH2OH group
Beta-anomers
Have the -OH on the anomeric carbon cis to the free -CH2OH group
Haworth projections
Provide a good way to represent three-dimensional structures
Mutarotation
“Cyclic compounds shift from one anomeric form to another with the straight-chain form as an intermediate
The hemiacetal ring of the sugar will break open spontaneously and then re-form. When the ring is broken, bond rotation occurs between C-1 and C-2 to produce either the alpha- or beta- anomer”
Monosaccharides
Single carbohydrate units, with glucose as the most commonly observed monomer
What is the most commonly observed monomer?
Glucose
What reactions can monosaccharides undergo?
Oxidation-reaction, esterification and glycoside formation
Oxidation of aldoses result in:
Aldonic acids
Reduction of aldoses result in:
Alditols
Reducing sugars
Sugars that are oxidized and act as reducing agents
How can reducing sugars be detected?
Tollen’s reagent or Benedict’s reagent
Deoxy sugars
Sugars with a -H replacing an -OH group
Esterification
Sugars can react with carboxylic acids and their derivatives, forming esters
Phosphorylation
“Similar to esterification
Phosphate ester is formed by transferring a phosphate group from ATP onto a sugar”
Glycoside formation
The basis for building complex carbohydrates and requires the anomeric carbon to link to another sugar
Disaccharides
Form as result of glycosidic bonding between two monosaccharide subunits
How do polysaccharides form?
By repeated monosaccharide or polysaccharide glycosidic bonding
Common disaccharides
Sucrose, lactose and maltose
Sucrose
Glucose-alpha-1,2-fructose
Lactose
Galactose-beta-1,4-glucose
Maltose
Glucose-alpha-1,4-glucose
Polysaccharides
Cellulose, starches and glycogen
Cellulose
The main structural component for plant cell walls and is a main source of fiber in the human diet
Starches subtypes
Amylose and amylopectin
Starches
Function as a main energy storage form for plants
Glycogen
Functions as a main energy storage form for animals
Aldohexose
Sugar that has one aldehyde group and six carbons, e.g. glucose
Is glucose an aldohexose?
Yes
How many chiral carbons does glucose have?
4
Glycosidation
The addition of a sugar to another compound
Tautomerization
A rearrangement of bonds to undergo keto-enol shifts
Anomerization
Refers to ring closure of a monosaccharide, creating an anomeric carbon
What happens when glucose reacts with ethanol with an acid catalyst?
The hemiacetal is converted to an acetal via replacement of the anomeric hydroxyl group with an an alkoxy group, the result is a type of acetal known as a glycoside
Beta-amylase
Cleaves amylose at the nonreducing end of the polymer to yield maltose
Alpha-amylase
Cleaves amylose anywhere along the chain to yield short polysaccharides, maltose and glucose
Debranching enzyme
Removes oligosaccharides from a branch in glycogen or starches
Glycogen phosphorylase
Yields glucose 1-phosphate (GMP)
How to convert a straight-chain Fischer projection into a chair/ring conformation?
- Draw out the Haworth conformation
- All the groups on the right in the Fischer projection will go on the bottom of the Haworth projection
- All the groups on the left in the Fischer projection will go on the top of the Haworth projection
- Draw out the chair structure, with the oxygen in the back right corner
- Label the carbons in the ring 1 through 5 from the oxygen and moving clockwise around the ring
- Draw in the lines for all the axial substituents, alternating above and below the ring
- What was pointing down in the Haworth projection, will also point down for the chair conformation
- What was pointing up in the Haworth projection, will also point up for the chair conformation
Which polysaccharides display branching structure?
Glycogen and amylopectin
What linkages does glycogen use?
Alpha-1,4 and alpha 1,6
What linkages does amylopectin use?
Alpha-1,4 and alpha 1,6
What linkages does cellulose use?
Beta-1,4
What linkages does amylose use?
Alpha-1,4
Can humans digest maltose?
Yes
Can humans digest cellobiose?
No
Why can’t humans digest cellobiose?
Because cellobiose contain beta-glycosidic linkages and humans do not have the enzymes to cleave them
What are the cyclic forms of monosaccharides?
Hemiacetals and hemiketals
Which components of membrane lipids contribute to their structural role in membranes?
The fatty acid tails form the bulk of the phospholipid bilayer and play a predominantly structural role
Which components of membrane lipids contribute to function?
“The polar head group determines the function of the membrane lipid due to its constant exposure to the exterior environment of the phospholipid bilayer
The degree of unsaturation of fatty acid tails can also play a small role in function”
What is the difference between a sphingolipid that is also a phospholipid and one that is not?
The difference is the bond between the sphingosine backbone and the head group. When this is a phosphodiester bond, it’s a phospholipid. Nonphospholipid sphingolipids include glycolipids, which contain a glycosidic linkage to a sugar.
Types of sphingolipids
Sphingomyelin, glycosphingolipid and ganglioside
What would happen if an amphipathic molecule were placed in a nonpolar solvent rather than an aqueous solution?
In a nonpolar solvent, we would see the opposite of what happens in a polar solvent like water: the hydrophilic, polar part of the molecules would be sequestered inside, while the nonpolar, hydrophobic part of the molecules would be found on the exterior and exposed to the solvent
Phospholipid structure
Phosphodiester bond between the sphingosine backbone and the head group
Are phospholipids sphingolipids?
Yes
Glycolipid structure
Glycosidic linkage between sphingosine backbone and a sugar
Are glycolipids sphingolipids?
Yes
Is sphingomyelin a phospholipid or a glycolipid?
Phospholipid
Is glycosphingolipid a phospholipid or a glycolipid?
Glycolipid
Is ganglioside a phospholipid or a glycolipid?
Glycolipid
What is the functional group of a sphingomyelin?
Phosphatidylethanolamine / phosphatidylcholine
What is the functional group of a glycosphingolipid?
Sugars (mono- or polysaccharides)
What is the functional group of a ganglioside?
Oligosaccharides and N-acetylneuraminic acid (NANA)
How many carbons are in a diterpene?
20
What is the difference between a steroid snd a steroid hormone?
"A steroid is defined by its structure: it includes 3 cyclohexane rings and one cyclopentane ring A steroid hormone is a molecule within this class that also functions as a hormone, meaning that it travels in the bloodstream, is active at low concentrations, has high-affinity reception and affects gene expression and metabolism."
NSAIDs block prostaglandin production in order to reduce pain and inflammation. What do prostaglandins do to bring about these symptoms?
Prostaglandins regulate the synthesis of cAMP, which is involved in many pathways, including those that drive pain and inflammation
Fat-soluble vitamins
Vitamin A (carotene), vitamin D (cholecalciferol), vitamin E (tocopherols) and vitamin K (phylloquinone and menaquinones)
Terpene unit
Made of 2 isoprene units
How many carbons are in an isoprene?
5
Vitamin A (carotene)
“Metabolized to retinal for vision
Metabolized to retinoid acid for gene expression in epithelial development”
Vitamin D (cholecalciferol)
Metabolized to calcitriol in the kidneys to regulate calcium and phosphate homeostasis in the intestines (increasing calcium and phosphate absorption) and promoting bone formation
Vitamin E (tocopherols)
Metabolized to biological antioxidants, using their aromatic rings to destroy free radicals, preventing oxidative damage
Vitamin K (phylloquinone and menaquinones)
“Important for the formation of prothrombin, a clotting factor
Performs posttranslational modification to a number of proteins, creating calcium-binding sites”
How does the human body store spare energy?
The human body stores energy as glycogen and triacylglycerols
Why doesn’t the human body store most energy as sugar?
Triacylglycerols are preferred because their carbons are more reduced, resulting in a larger amount of energy yield per unit weight. In addition, due to their hydrophobic nature, triacylglycerols do no need to carry extra weight from hydration.
Describe the structure of triacylglycerols (triglycerides)
One glycerol attached to three fatty acids by ester bonds
The carbon atoms in lipids are more reduced than those in carbohydrates, giving them twice as pure energy per gram during oxidation
Hydrophobic, so they are not hydrated by body water and do not carry additional water weight
What bonds are broken during saponification?
The ester bonds of triacylglycerols (triglycerides) are broken to form a glycerol molecule and the salts of fatty acids (soap)
Why does soap appear to dissolve in water, and how is this fact important to cleaning?
Soap appears to dissolve in water because amphipathic free fatty acid salts form micelles, with hydrophobic fatty acid tails towards the center and carboxylate groups facing outward towards the water. Fat-soluble particles can then dissolve inside micelles in the soap-water solution and wash away. Water-soluble compounds can freely dissolve in the water.
What are triacylglycerols (triglycerides) used for?
Energy storage
Are lipids soluble in water?
No
Are lipids soluble in nonpolar organic solvents?
Yes
Phospholipids
“Amphipathic
Form the bilateral of biological membrane
Contain a hydrophilic (polar) head group and hydrophobic (nonpolar) tails
The head group is attacked by a phosphodiester linkage”
Are saturated fatty acids more or less fluid than unsaturated fatty acids?
Less
Glycerophospholipids
Phospholipids that contain a glycerol backbone
Sphingolipids
Contain a sphingosine or sphenoid backbone
Can have a phosphodiester bond (phospholipids) or glycosidic bonds (glycolipids)
Used in ABO blood typing
Sphingophospholipids
Sphingolipids that are phospholipids because they have a phosphodiester bond
Sphingomyelins
"A major class of sphingophospholipids and contain a phosphatidylcholine or phosphatidylethanolamine head group A major component of the myelin sheath"
Glycosphingolipids
Attached to sugar moieties instead of a phosphate group
Cerebrosides
Have one sugar connected to a sphingosine
Globosides
Have two or more sugars connected to a sphingosine
Gangliosides
Contain oligosaccharides with at least one terminal N-acetylneuraminic acid (NANA) also called sialic acid
Waxes
Contain long-chain fatty acids esterified to long-chain alcohols
Used as protection against evaporation (dehydration) and parasites in plants and animals
Generally have melting points above room temperature
Terpenes
Odiferous steroid precursors made from isoprene
Isoprene
A five-carbon molecule
Monoterpene
“One terpene unit
Contains two isoprene units”
Terpenoids
“Derived from terpenes via oxygenation or backbone rearrangement
They have similar odorous characteristics”
Steroids
Contain three cyclohexane rings and one cyclopentane ring
Steroid hormones
Have high-affinity receptors, work at low concentrations, travel in the bloodstream from endocrine glands and affect gene expression and metabolism by binding to DNA as part of the hormone-receptor complex, but not by itself
Cholesterol
A steroid important to membrane fluidity and stability; it serves as a precursor to a host of other molecules like steroid hormones
Prostaglandins
Autocrine and paracrine signals molecules that regulate cAMP levels
Have powerful effects on smooth muscle contraction, body temperature, the sleep-wake cycle, fever, inflammation and pain
Their synthesis is inhibited by NSAIDs
What causes rickets?
Vitamin D (cholecalciferol) deficiency
What is the preferred method of string energy for long-term use?
Triacylglycerols (triglycerides)
Adipocytes
Animal cells specifically used for storage of large triacylglycerol deposits
Free fatty acids
Unesterified fatty acids that travel in the bloodstream
Soap
Salts of free fatty acids
Acts as surfactants, forming micelles
How is soap synthesized?
Through saponification
Saponification
The ester hydrolysis of triacylglycerols using a strong base, like NaOH or KOH
Micelle
Can dissolve a lipid-soluble molecule in its fatty acid core
Washes away with water because of its shell of carboxylate head groups
Collection of lipids with their hydrophobic ends oriented towards the center and their charged ends orientated towards the aqueous environment
Collect lipids within their hydrophobic centers
Types of glycolipids
Cerebroside, globoside and ganglioside
What kind of bond connects the head group of a sphingomyelin to its backbone?
Phosphodiester bond
Saturation
Describes the number of double or triple bonds in a fatty acid tail
Determines membrane fluidity
Are more saturated fatty acids make for a more or less fluid of solution?
Less
Fully saturated fatty acids
Only have single bonds
Are glycerophospholipids a subtype of sphingolipids?
No
Nucleoside
Contains a five-carbon sugar (pentose) and a nitrogenous base
Nucleotide
Composed of a nucleoside plus one to three phosphate groups
What are the base-pairing rules according to the Watson-Crick model?
A pairs with T in DNA, using 2 hydrogen bonds
A pairs with U in RNA, using 2 hydrogen bonds
C pairs with G in DNA and RNA, using 3 hydrogen bonds
The backbone is composed of alternating sugar and phosphate groups, and is always read 5’ to 3’
The two strands are antiparallel in polarity and are wound into a double helix
What are the three major structural differences between DNA and RNA?
DNA contains deoxyribose, while RNA contains ribose
DNA contains thymine, while RNA contains uracil
DNA is double-stranded, while RNS is single-stranded
How does the aromaticity of purines and pyrimidines underscore their genetic function?
The aromaticity of nucleic acids makes these compounds very stable and interactive
Stability is important for storing genetic information and avoiding spontaneous mutations
What are the five histone proteins in eukaryotic cells?
H1, H2A, H2B, H3, H4
Which histone is not part of the histone core around which DNA wraps to form chromatin?
H1
What is the density of chromatin packing in heterochromatin?
Dense
What is the density of chromatin packing in euchromatin?
Uncondensed (not dense)
What is the appearance of heterochromatin under light microscopy?
Dark
What is the appearance of euchromatin under light microscopy?
Light
What is the transcriptional activity of heterochromatin?
Silent
What is the transcriptional activity of euchromatin?
Active
What property of telomeres and centromeres allows them to stay tightly raveled, even when the rest of DNA is uncondensed?
High GC-content increases hydrogen bonding, making the association between DNA strands very strong at telomeres and centromeres
Is helicase present in prokaryotes or eukaryotes?
Both
Helicase
Unwinds DNA double helix
Is single-stranded DNA-binding protein present in prokaryotes or eukaryotes?
Both
Single-stranded DNA-binding protein
Prevents reannealing of DNA double helix during replication
Is primase present in prokaryotes or eukaryotes?
Both
Primase
Places ~10-nucleotide RNS primer to begin DNA replication
Is DNA polymerase III present in prokaryotes or eukaryotes?
Prokaryotes
DNA polymerase III
Adds nucleotides to growing daughter strand
Is DNA polymerase alpha present in prokaryotes or eukaryotes?
Eukaryotes
DNA polymerase alpha
Adds nucleotides to growing daughter strand
Is DNA polymerase I present in prokaryotes or eukaryotes?
Prokaryotes
DNA polymerase I
Fills in gaps left behind after RNS primer excision
Is RNase H present in prokaryotes or eukaryotes?
Eukaryotes
RNase H
Excises RNA primer
Is DNA ligase present in prokaryotes or eukaryotes?
Both
DNA ligase
Joins DNA strands (especially between Okazaki fragments)
Is DNA topoisomerases present in prokaryotes or eukaryotes?
Both
DNA topoisomerases
Reduces torsional strain from positive supercoils by introducing nicks in DNA strand
Involved in DNA replication and transcription
Which is more prone to mutations: the leading strand or lagging strand?
The lagging strand, because it must constantly start and stop the process of DNA replication. Additionally, it contains many more RNA primers, all of which must be removed and filled in with DNA
Telomere
The ends of eukaryotic chromosomes
Contain repetitive sequences of noncoding DNA
Protect the chromosome from losing important genes from the incomplete replication of the 5’ end of the DNA strand
Oncogene (porto-oncogenes)
Code for cell cycle-promoting proteins; when mutated, a porto-oncogene becomes an oncogene, promoting rapid cell cycling
(stepping on the gas pedal)
Results in cancer through gain of function mutations
Tumor supressor gene
Code for repair or cell-cycle inhibiting proteins; when mutated, the cell cycle is allowed to proceed unchecked
(cutting the brakes)
Results in cancer through loss of function mutations
How does DNA polymerase recognize which strand is the template strand once the daughter strand is synthesized?
The parent strand is more heavily methylated, whereas the daughter strand is bare methylated at all
When does DNA polymerase (proofreading) happen in the cell cycle?
S
When does mismatch repair happen in the cell cycle?
G2
When does nucleotide excision repair happen in the cell cycle?
G1 and G2
When does base excision repair happen in the cell cycle?
G1 and G2
Which enzymes or genes work during DNA polymerase (proofreading)?
DNA polymerase
Which enzymes or genes work during mismatch repair?
MSH2, MLH1 (MutS and MutL in prokaryotes)
Which enzymes or genes work during nucleotide excision repair?
Excision endonuclease
Which enzymes or genes work during base excision repair?
Glycosylase, AP endonuclease
What is the key structural difference in the types of lesions corrected by nucleotide excision repair v. those corrected by base excision repair?
Nucleotide excision repair corrects lesions that are large enough to distort the double helix
Base excision repair corrects lesions that are small enough not to distort the double helix
Genomic library
Include all of the DNA in an organism’s genome, including noncoding regions
Useful for studying DNA in introns, centromeres or telomeres
cDNA library (expression libraries)
Only include expressed genes from a given tissue, but can be used to express recombinant proteins or to perform gene therapy
Contains only the exons of genes that are transcriptionally active in the sample tissue
Polymerase chain reaction (PCR)
Increases the number of copies of a given DNA sequence and can be used for a sample containing very few copies of the DNA sequence
Used to clone a sequence of DNA using a DNA sample, a primer, free nucleotides and enzymes
The polymerase from Thermus aquatics is used because the reaction is regulated by thermal cycling, which would denature human enzymes
A primer must be prepared with a complementary sequence to part of the DNA of interest
Repeated heating and cooling cycles allow the enzymes to act specifically and replaces helicase
Each cycle of the polymerase chain reaction doubles the amount of DNA of interest
Southern blotting
Useful when searching for a particular DNA sequence because it separates DNA fragments by length and then probes for a sequence of interest
During DNA sequencing, why does the DNA polymer stop growing once a dideoxyribonucleotide is added?
Dideoxyribonucleotides lack the 3’-OH group that is required for DNA strand elongation. Thus, once a dideoxyribonucleotide is added to a growing DNA molecule, no more nucleotides can be added because dideoxyribonucleotides have no 3/-OH group with which to form a bond.
What is the difference between a transgenic and a knockout mouse?
Transgenic mice have a gene introduced into their germ line or embryonic stem cells to look at the effects of that gene; they are therefore best suited for studying the effects of dominant alleles. Knockout mice are those in which a gene of interest has been removed, rather than added.
Deoxyribonucleic acid (DNA)
A macromolecule that stores genetic information in all living organisms
What kind of sugar is in DNA?
Deoxyribose
What kind of sugar is in RNA?
Ribose
Nucleotides in RNA and DNA
Adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U)
Purines
Adenine (A) and guanine (G)
Pyrimidines
Cytosine (C), thymine (T) and uracil (U)
Does RNA contain thymine (T)?
No, it contains uracil (U) instead
Aromatic compounds
Cyclic, planar, conjugated and contain 4n + 2 pi electrons (where n = any integer; Huckel’s rule)
Heterocycles
Ring structures that contain at least two different elements in the ring
Chargaff’s rules
Purines and pyrimidines are equal in number in a DNA molecule, and that because of base-pairing, the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine
Most DNA is what kind of DNA?
B-DNA (i.e. right-handed helix)
Z-DNA
Zigzag shaped
Present in low concentrations
Seen with high GC-content or high salt concentrations
B-DNA
Right-handed helix
Denaturation
Pulling apart DNA strands
Reannealation
Bringing back DNA strands together
Factors aiding denaturation
Heat, alkaline pH, chemicals like formaldehyde and urea
How many chromosomes are in a human cell?
46
Which histone proteins form nucleosomes?
H2A, H2B, H3 and H4
Which histone stabilizes the nucleosome?
H1
Chromatin
DNA with its associated histones
Heterochromatin
Dense, transcriptionally silent DNA that appears dark under light microscopy
Euchromatin
Less dense, transcriptionally active DNA that appears light under light microscopy
Telomeres
The ends of chromosomes
Contain high GC-contain to precent unraveling of the DNA
During replication, they are slightly shortened, although this can be (partially) reversed by the enzyme telomerase
Centromeres
Located in the middle of chromosomes and hold sister chromatids together until they are separated during anaphase in mitosis
They contain a high GC-contain to maintain a strong bond between chromatids
Replisome (replication complex)
A set of specialized proteins that assist the DNA polymerases
DNA replication
- Helicases unwind the DNA at the origin of replication, producing two replication forks on either side of the origin
- Unwound strands are kept from reanneling or being degraded by single-stranded DNA-binding proteins
- Supercoiling causes torsional strain on the DNA molecule, which can be released by DNA topoisomerases, with creates nicks in the DNA molecule
- DNA cannot be synthesized without an adjacent nucleotide to hook onto, so a small RNA primer is put down by primase
- DNA polymerase III (prokaryotes) or DNA polymerase alpha, delta and epsilon (eukaryotes) can then synthesize a new strand of DNA; they read the template DNA 3’ tp 5’ and synthesize the new strand 5’ to 3’
- RNA primers can be removed by DNA polymerase I (prokaryotes) or RNase H (eukaryotes), and filled in with DNA by DNA polymerase I (prokaryotes or DNA polymerase delta (eukaryotes)
- DNA ligase can then fuse the DNA strands together to create one complete molecule
How many origins or replication does the prokaryotic chromosome have?
One
How many origins or replication does the eukaryotic chromosome have?
Many
Is DNA replication conservative, semiconservative or nonconservative?
Semiconservative: one old parent strand and one new daughter strand is incorporated into each of the two new DNA molecules
Leading strand
Requires only one primer and can then be synthesized continuously in its entirety
Lagging strand
Requires many primers and is synthesized in discrete sections called Okazaki fragments
Cancer
Unchecked cell proliferation with the ability to spread by local invasion or metastasize (migrate to distant sites via the bloodstream or lymphatic system)
DNA polymerase proofreading
Occurs during replication
Excises incorrectly matched bases
Mismatch repair
Occurs during G2 phase of the cell cycle
Uses genes MSH2 and MLH1
Nucleotide excision repair
Fixes helix-deforming lesions of DNA (such as thymine dimers) via a cut-and-patch process that requires an excision endonuclease
Base excision repair
Fixes nondeforming lesions of the DNA helix (such as cytosine deamination) by removing the base, leaving an apurinic/apyrimidinic (AP) site. An Ap endonuclease then removes the damaged sequence, which can be filled in with the correct bases.
Recombinant DNA
DNA composed of nucleotides from two different sources
DNA cloning
introduces a fragment of DNA into a vector plasmid
Restriction enzyme (restriction endonuclease)
Cuts both the plasmid and the fragment, which are left with sticky ends
Once the fragment binds to the plasmid, it can be introduced into a bacterial cell and permitted to replicate, generating many copies of the fragment of interest
Vectors contain an origin of replication, the fragment of interest, and at least one gene for antibiotic resistance (to permit for selection of that colony after replication)
Once replicated, the bacterial cells can be used to create a protein of interest from the vector
Hybridization
The joining of complementary base pair sequences
How can DNA molecules be separated?
By size, using agarose gel electrophoresis
Gene therapy
A method of curing genetic deficiencies by introducing a functional gene with a viral vector
Transgenic mice
Created by integrating a gene of interest into the germ line or embryonic stem cells of a developing mouse
Chimeras
Organisms that contain cells from two different lineages
Knockout mice
Created by deleting a gene of interest
What can be detected with southern blotting?
DNA
What can be detected with northern blotting?
mRNA (ss)
What can be detected with western blotting?
Proteins
Conjugation
Every atom in the ring must have at least one unhybridized p-orbital (alternating double or triple bonds)
cDNA (complementary DNA)
Results from the reverse transcription of processed mRNA
What is restriction endonuclease used for?
Gene therapy, Southern blotting and DNA repair
Why might uracil be excluded from DNA but not RNA?
Cytosine degradation results in uracil
One common DNA mutation is the transition from cytosine to uracil in the presence of heat. DNA repair enzymes recognize uracil and correct this error by excising the base and inserting cytosine. RNA exists only transiently in the cell, such that cytosine degradation is insignificant. Were uracil to be used in DNA under normal circumstances, it would be impossible to tell if a base should be uracil or if it is a damaged cytosine nucleotide.
mRNA (messenger RNA)
Carries information from DNA by traveling from the nucleus (where it is transcribed) to the cytoplasm (where it is translated)
tRNA (transfer RNA)
Translates nucleic acids to amino acids by pairing its anticodon with mRNA codons
It is charged with an amino acid, which can be added to the growing peptide chain
rRNA (ribosomal RNA)
Forms much of the structural and catalytic component of the ribosome, and acts as a ribozyme to create peptide bonds between amino acids
Which mRNA codon is the start codon?
AUG
Which amino acid does the start codon for?
Methionine
Which mRNA codons are the stop codons?
UAA, UGA and UAG
Wobble
Refers to the fact that the third base in a codon often plays no role in determining which amino acid is translated from that codon. This is protective because mutations in the wobble position will not have any effect on the protein translated from that gene.
What change in DNA sequence does a silent (degenerate) mutation bring?
Substitution of bases in the wobble position, introns or noncoding DNA
What change in DNA sequence does a missense mutation bring?
Substitution of one base, creating an mRNA codon that matches a different amino acid
What change in DNA sequence does a nonsense mutation bring?
Substitution of one base, creating a stop codon
What change in DNA sequence does a frameshift mutation bring?
Insertion or deletion of bases, creating a shift in the reading frame of the mRNA
What effect on the encoded proton does a silent (degenerate) mutation have?
No change is observed
What effect on the encoded proton does a missense mutation have?
One amino acid is changed in the protein; variable effects on function depending on specific change
What effect on the encoded proton does a nonsense mutation have?
Early truncation of protein; variable effects on function, but usually more severe than missense mutations
What effect on the encoded proton does a frameshift mutation have?
Change in most amino acids after the site of insertion deletion; usually the most severe of the types of mutations
RNA polymerase I in eukaryotic cells
Synthesizes most rRNA
RNA polymerase II in eukaryotic cells
Synthesizes mRNA (hnRNA) and snRNA
Binds to the TATA box within the promoter region of the gene (25 base pairs upstream from the first transcribed base)
RNA polymerase III in eukaryotic cells
Synthesizes tRNA and some rRNA
When starting transcription, where does RNA polymerase bind?
RNA polymerase II binds to the TATA box, which is located within the promoter region of a relevant gene, at about -25
What are the three major posttranscriptional modifications that turn hnRNA into mature mRNA?
- Splicing: removal of introns, joining of exons :: Uses snRNA and snRNPs in the spliceosome to create a lariat (made of introns), which is then degraded. Exons are ligated together
- 5’ cap: addition of a 7-methylguanylate triphosphate cap to the 5’ end of the transcript
- 3’ poly-A tail: addition of adenosine bases to the 3’ end to protect against degradation
Alternative splicing
The ability of some genes to use various combinations of exons to create multiple proteins from one hnRNA transcript. This increases protein diversity and allows a species to maximize the number of proteins it can create from a limited number of genes
What are the three steps of translation?
Initiation, elongation and termination
A site in a ribosome
Binds incoming aminoacyl-tRNA using codon-anticodon pairing
P site in a ribosome
Holds growing polypeptide until peptide transferase forms peptide bond and polypeptide is handed to A site
E site in a ribosome
Transiently holds uncharged tRNA as it exits the ribosome
What are the major posttranslational modifications that occur in proteins?
Proper folding by chaperones, formation of quaternary structure, cleavage of proteins or signal sequences, and covalent addition of other biomolecules (phosphorylation, carboxylation, glycosylation, prenylation)
trp operon
Negative repressible system
lac operon
Negative inducible system
What are the components of the operon from 5’ to 3’?
Regulator gene, promoter site, operator site, structural gene
Positive control system
Require the binding of a protein to the operator site to increase transcription
Negative control system
Require the binding of a protein to the operator site to decrease transcription
Regulator gene
Transcribed to form repressor protein
Promoter site
Site of RNA polymerase binding (similar to promoters in eukaryotes)
Operator site
Binding site for repressor protein
Structural gene
The gene of interest; its transcription is dependent on the repressor being absent from the operator site
In an enhancer, what are the differences between signal molecules, transcription factors, and response elements?
Signal molecules include steroid hormones and second messengers, which kind to their receptors in the nucleus. These receptors are transcription factors that use their DNA-binding domain to attach to a particular sequence in DNA called a response element. Once bonded to the response element, these transcription factors can then promote increased expression of the relevant gene.
By what histone and DNA modifications can genes be silenced in eukaryotic cells? Would these processes increase the proportion of heterochromatin or euchromatin?
Histone deacetylation and DNA methylation will both down regulate the transcription of a gene. These processes allow the relevant DNA to be clumped more tightly, increasing the proportion of heterochromatin.
Central dogma
DNA is transcribed to RNA, which is translated to protein
Point mutations
Silent, nonsense (truncation) and missense mutations
Silent mutation
Have no effect on protein synthesis
Nonsense (truncation) mutation
Produce a premature stop codon
Missense mutation
Produce a codon that codes for a different amino acid
Frameshift mutation
Result from nucleotide addition or deletion
Change the reading frame of subsequent codons
Types of RNA
Messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA)
hnRNA
Synthesized from the DNA template (antisense) strand
How can prokaryotic cells increase the variability of gene products from one transcript?
Polycistronic genes :: starting transcription in different sites within the gene, leading to different gene products
How can eukaryotic cells increase the variability of gene products?
Alternative splicing :: combining different exons in a modular fashion to acquire different gene products
Initiation in translation (prokaryotes)
Occurs when the 30S ribosome attaches to the Shine-Dalgarno sequence and scans for a start codon; it lays down N-formylmethionine in the P site of the ribosome
Initiation in translation (eukaryotes)
Occurs when the 40S ribosome attaches to the 5’ cap and scans for a start codon; it lays down methionine in the P site of the ribosome
Elongation in translation
Involves the addition of a new aminoacyl-tRNA into the A site of the ribosome and transfer of the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site. The now uncharged tRNA pauses in the E site before exiting the ribosome
Termination in translation
Occurs when the codon in the A site is a stop codon; release factor places a water molecule on the polypeptide chain and thus releases the protein
Jacob-Monod model
Explains how operons work
Operons
Inducible or repressible clusters of genes transcribed as a single mRNA
Inducible system
e.g. lac operon
Bonded to a repressor under normal conditions
Can be turned on by an inducer pulling the repressor from the operator site
Repressible system
e.g. trp operon
Transcribed under normal conditions
Can be tuned off by a corepressor coupling with a repressor and the binding of this complex to the operator site
Transcription factors
Search for promoter and enhancer regions in the DNA
Promoters
Within 25 base pairs of the transcription start site
Enhancers
More than 25 base pairs away from the transcription start site
Transcriptional regulatory sequences that function by enhancing the activity of RNA polymerase at a single promoter site
Does histone acetylation incense or decrease the ability of transcriptional enzymes to access the DNA?
Increase
Does DNA methylation incense or decrease the ability of transcriptional enzymes to access the DNA?
Decrease
What role does peptide transferase play in protein synthesis?
It catalyzes the formation of a peptide bond between the incoming amino acid in the A site and the growing polypeptide chain in the P site
What is the role of initiation and elongation factors?
Transporting charged tRNA molecules into the ribosome and advancing the ribosome down the mRNA transcript
Chaperones
Maintain a protein’s 3D shape as it is formed
Which stage of protein synthesis (initiation, elongation and termination) does not require energy?
None : They all require energy
When trypsin converts chymotrypsinogen to chymotrypsin, some molecules of chymotrypsin bind to a repressor, which in turn binds ti an operator region and prevents further transcription of trypsin. This is most similar to:
a) trp operon during lack of tryptophan
b) trp operon during abundance of tryptophan
c) lac operon during lack of lactose
d) lac operon during abundance of lactose
trp operon during abundance of tryptophan
Which RNA molecule or protein is not found in the spliceosome during intron excision?
a) snRNA
b) hnRNA
c) shRNA
d) snRNPs
shRNA (short hairpin RNA)
It is a useful in biotechnology tool using in RNA interference. It is not, however, produced in the nucleus for use in the spliceosome. It targets mRNA to be degraded in the cytoplasm; it is not utilized in splicing of the hnRNA (heterogeneous nuclear RNA) and snRNPs (small nuclear ribonucleoproteins), however, do bind to the hnRNA to induce splicing.
A eukaryotic cell has been found to exhibit a truncation mutation that creates an inactive RNA polymerase I enzyme. Which type of RNA will be affected by this inactivation?
rRNA
RNA polymerase I in eukaryotes is found in the nucleolus and is in charge of transcribing most of the rRNA for use during ribosomal creation. RNA polymerase II is responsible for hnRNA and snRNA. RNA polymerase III is responsible for tRNA and the 5S rRNA.
Double-stranded RNA cannot be translated by the ribosome and is marked for degradation in the cell. Which of the following strands of RNA would prevent mature mRNA in the cytoplasm from being transcribed?
a) Identical mRNA to the one produced
b) Antisense mRNA to the one produced
c) mRNA with thymine substituted for uracil
d) Sense mRNA to the one produced
Antisense mRNA to the one produced
The mRNA produced has the same structure as the sense strand of DNA (with uracils instead of thymines). Because bonding of nucleic acids is always complementary but antiparallel, the antisense strand of mRNA would be the one that binds to the produced mRNA, creating double-stranded RNA that is then degraded once found in the cytoplasm.
Flippases
Responsible for the movement of phospholipids between the layers of the plasma membrane because it is otherwise energetically unfavorable
Specific membrane proteins that maintain the bidirectional transport of lipids between the layers of the phospholipid bilayer in cells
Lipid rafts
Aggregates of specific lipids in the membrane that function as attachment points for other biomolecules and play roles in signaling
Order membrane components from most plentiful to least plentiful
- Lipids (including phospholipids and cholesterol)
- Proteins (including transmembrane proteins [channels and receptors], membrane associated proteins, and embedded proteins)
- Carbohydrates (including the glycoprotein coat and signaling molecules)
- Nucleic acids
How does cholesterol play a role in the fluidity and stability of the plasma membrane?
Moderates membrane fluidity by interfering with the crystal structure of the cell membrane and occupying space between phospholipid molecules at low temperatures, and by restricting excessive movement of phospholipids at high temperatures
Provides stability by cross-linking adjacent phospholipids through interactions at the polar head group and hydrophobic interactions at the nearby fatty acid tail
What are the three classes of membrane proteins?
Transmembrane proteins, embedded membrane proteins, membrane associated (peripheral) proteins
Transmembrane proteins
Serve as channels or receptors
Can have one or more hydrophobic domains
Embedded membrane proteins
Have catalytic activity linked to nearby enzymes
Cellular communication
Membrane associated (peripheral) proteins
Involved in signaling or are recognition molecules on the extracellular surface
Gap junctions
Allow for the intercellular transport of materials between adjacent cells and do not prevent paracellular transport of materials
Exist in discontinuous bunches around the cell
Tight junctions
Not used for intercellular transport but do prevent paracellular transport
Form bands around the cell
What is the primary thermodynamic factor responsible for passive transport?
Entropy
What is the relationship between osmotic pressure and the direction of osmosis through a semipermeable membrane?
As osmotic pressure increases, more water will tend to follow into the compartment to decrease solute concentration
Osmotic pressure is often considered a “sucking” pressure because water will move towards the compartment with the highest osmotic pressure
Types of active transport
Primary active transport and secondary active transport
Primary active transport
Uses ATP as an energy source for the movement of molecules against their concentration gradient
Secondary active transport
Uses an electrochemical gradient to power the transport
Symport
Moves both particles in secondary active transport across the membrane in the same direction
Antiport
Moves particles in secondary active transport across the membrane in opposite directions
Types of secondary active transport
Symport and antiport
How is the resting membrane potential maintained?
Sodium-potassium pumps and leak channels
What distinguishes the inner mitochondrial membrane from other biological membranes?
The inner mitochondrial membrane lacks cholesterol
What is the pH gradient between the cytoplasm and the intermembrane space of the mitochondrion?
There is no pH gradient between the cytoplasm and the intermembrane space because the outer mitochondrial membrane has such high permeability to biomolecules (the potion-motive force of the mitochondria is across the inner mitochondrial membrane, not the other mitochondrial membrane)
Osmotic pressure (pi)
= i M R T
Nernst equation
(E) = RT / zF ln ([ion]outside / [ion]inside) = 61.5 / z log ([ion]outside / [ion]inside)
Calculates the electrical potential created by one ion
Goldman-Hodgkin-Katz voltage equation
Vm = 61.5 log ([{P Na+ x [Na+]outside} + {P K+ x [K+]outside} + {P Cl- x [Cl-]inside}] / [{P Na+ x [Na+]inside} + {P K+ x [K+]inside} + {P Cl- x [Cl-]outside}])
Calculates the resting membrane potential at physiological temperature
Derived from the Nernst equation
Fluid mosaic model
Accounts for the presence of lipids, proteins, and carbohydrates in a dynamic, semisolid plasma membrane that surrounds cells
Is the cell membrane fluid or static?
Fluid
Lipids move freely in the plane of the cell membrane and can assemble into:
lipid rafts
Can proteins and carbohydrates also move within the cell membrane?
Yes, but their movement is slowed by their relatively large size
What is the primary cell membrane component?
Lipids
Triacylglycerols
Phospholipid precursors
Found in low levels in the cell membrane
Free fatty acids
Phospholipid precursors
Found in low levels in the cell membrane
Glycerophospholipids
Replace one fatty acid with a phosphate group, which is often linked to other hydrophilic groups
Cholesterol
Present in large amounts in the cell membrane and contributes to membrane fluidity and stability
Waxes
Present in small amounts in the cell membrane (if at all) and are Mose prevalent in plants and function in waterproofing and defense
Proteins in the cell membrane
Act as transporters, cell adhesion molecules and enzymes
Carbohydrates on the cell membrane
Form a protective glycoprotein coat
Function in cell recognition
Extracellular ligands
Bind to membrane receptors, which function as channels or enzymes in second messengers pathways
Cell-cell junctions
Cap, tight and desmosomes and hemidesmosomes
Desmosomes and hemidesmosomes
Anchor layers of epithelial tissue together
Concentration gradients
Help to determine appropriate membrane transport mechanisms in cells
Osmotic pressure
A colligative property
The pressure applied to a pure solvent to prevent osmosis and is used to express the concentration of the solution
Conceptualized as a sucking pressure in which a solution is drawing water in, proportional to its concentration
Passive transport
Does not require energy because the molecule is moving down its concentration gradient or from an area with higher concentration to an area with lower concentration
Types of passive transport
Simple, osmosis and facilitated
Simile diffusion
Does not require a transported
Small, non polar molecules passively move from an area of high concentration to an area of low concentration until equilibrium is achieved
Osmosis
The diffusion of water across a selectively permeable membrane
Facilitated diffusion
Uses transport proteins to move impermeable solutes across the cell membrane
Active transport
Requires energy in the form of ATP or an existing favorable ion gradient
Methods of engulfing material into cells or releasing material to the exterior cells via the cell membrane
Endocytosis and exocytosis
Pinocytosis
The ingestion of liquid into the cell in vesicles formed from the cell membrane
Phagocytosis
The ingestion of large, solid molecules into the cell in vesicles formed from the cell membrane
What causes the cell membrane potential to exist?
A difference in the number of positive and negative charges on either side of the membrane
How does the sodium-potassium pump maintain the membrane potential?
By moving three sodium ions out of the cell for every two potassium ions pumped in
How do leak channels maintain the membrane potential?
By allowing the passive transport of ions
How does the mitochondrial membrane differ from the cell membrane?
- The outer mitochondrial membrane is highly permeably to metabolic molecules and small proteins
- The inner mitochondrial membrane surrounds the mitochondrial matrix, where the citric acid cycle produces electrons used in the electron transport chain and where many other enzymes important in cellular respiration are located
- The inner mitochondrial membrane does not contain cholesterol
A student is trying to determine the type of membrane transport occurring in a cell. She find that the molecule to be transported is very large and pole, and when transported across the membrane, no energy is required. What is the most likely mechanism of transport?
Facilitated diffusion
A researcher treats a solution containing animal cells with ouabain, a poisonous substance that interferes with the sodium-potassium ATPase embedded in the cell membrane, and the cell lyses as a result. Which of the follow statements best describes ouabain’s effects?
a. Treatment with ouabain results in high levels of extracellular calcium
b. Treatment with ouabain results in high levels of extracellular potassium and sodium
c. Treatment with ouabain increases intracellular concentrations of sodium
d. Treatment with ouabain decreases intracellular concentrations of sodium
Treatment with ouabain increases intracellular concentrations of sodium
What does resting membrane potential depend on?
The differential distribution ions across the membrane
Active transport processes
Selective permeability of the phospholipid bilayer
Functions of the cell membrane
Cytoskeletal attachment, transport regulation, and second messenger reservoir
Where does protein synthesis occur?
In ribosomes
The dynamic properties of molecules in the cell membrane are most rapid in:
Phospholipids moving within the plane of the membrane
Movement of individual molecules in the cell membrane will be affected by size and polarity, just as with diffusion
Lipids are much smaller than proteins in the plasma membrane and will more more quickly
Lipids will move fastest within the plane of the cell membrane because the polar head group does not need to pass through the hydrophobic tail region in the same way that it would if it were moving between the membrane layers
A membrane receptor is most likely to be a(n):
Transmembrane protein with catalytic activity
Plasmodesmata
Cell-cell junctions that are found in plants, not animals
What is common between diffusion and osmosis?
They both rely on the electrochemical gradient of only the compound of interest
Is it possible for a cell to have a resting membrane potential of 0?
No, because the resting membrane potential creates a state that is capable of responding to stimuli. Signaling molecules and channels would not be useful with a membrane potential of zero.
In which tissues does GLUT 2 work?
Liver and pancreas
In which tissues does GLUT 4 work?
Adipose tissue and muscle
What is the Km of GLUT 2?
High (~15 mM)
What is the Km of GLUT 4?
Low (~5 mM)
Is GLUT 2 saturated at normal glucose levels?
No
It cannot be saturated under normal physiological conditions
Is GLUT 4 saturated at normal glucose levels?
Yes
It is saturated when glucose levels are only slightly above 5 mM
Is GLUT 2 responsive to insulin?
No, but it serves as glucose sensor to cause release of insulin in pancreatic beta-cells
Is GLUT 4 responsive to insulin?
Yes
How does insulin promote glucose entry into cells?
GLUT 4 is saturated when glucose levels are only slightly above 5 mM, so glucose entry can only be increased by increasing the number of transporters. Insulin promotes the fusion of vesicles containing preformed GLUT 4 with the cell membrane
What is the function of hexokinase?
Glucose –(phosphorylation)–> glucose 6-phosphate (G6P)
Traps glucose in the cell
How is hexokinase regulated?
Inhibited by glucose 6-phosphate (G6P)
Is the reaction involving hexokinase reversible?
No
What is the function of glucokinase?
Glucose –(phosphorylation)–> glucose 6-phosphate (G6P)
Traps glucose in the cell
Only works in the liver and pancreas
Works with GLUT 2 as part of the glucose sensor in beta-islet cells
How is glucokinase regulated?
Inhibited by insulin the liver
Is the reaction involving glucokinase reversible?
No
What is the function of phosphofructokinase-1 (PFK-1)?
Fructose 6-phosphate (F6P) –(phosphorylation + ATP)–> fructose 1,6-bisphosphate (F1,6BP)
Rate-limiting step in glycolysis
How is phosphofructokinase-1 (PFK-1) regulated?
Inhibited by ATP, citrate and glucagon
Activated by AMP, fructose 2,6-bisphosphate (F2,6BP) and insulin
Is the reaction involving phosphofructokinase-1 (PFK-1) reversible?
No
What is the function of glyceraldehyde-3-phosphate dehydrogenase (G3PD)?
Glyceraldehyde 3-phosphate (G3P) –(phosphorylation)–> 1,3-bisphosphoglycerate (1,3BPG) + NADH
Is the reaction involving glyceraldehyde-3-phosphate dehydrogenase (G3PD) reversible?
Yes
What is the function of 3-phosphoglycerate kinase?
1,3-bisphosphoglycerate (1,3BPG) + ADP –(substrate-level phosphorylation)–> 3-phosphoglycerate (3PG) + ATP
Is the reaction involving 3-phosphoglycerate kinase reversible?
Yes
What is the function of pyruvate kinase?
Phosphoenolpyruvate (PEP) + ADP (substrate-level phosphorylation)–> pyruvate + ATP
How is pyruvate kinase regulated?
Activated by fructose 1,6-bisphosphate (F1,6BP)
Is the reaction involving pyruvate kinase reversible?
No
Why must pyruvate undergo fermentation for glycolysis to continue?
Fermentation must occur to regenerate NAD+, which is in limited supply in cells. Fermentation generates no ATP or energy carriers; it merely regenerates the coenzymes needed in glycolysis
Why is it necessary that fetal hemoglobin does not bind 2,3-BPG?
The binding of 2,3-BPG decreases hemoglobin’s affinity for oxygen. Fetal hemoglobin must be able to steal oxygen from maternal hemoglobin at the placental interface; therefore, it would be disadvantageous to lower its affinity for oxygen
Which enzyme is responsible for trapping galactose in the cell?
Galactokinase
Which enzyme in galactose metabolism results in a product that can feed directly into glycolysis, linking the two pathways?
Galactose-1-phosphate uridyltransferase
Which enzyme is responsible for trapping fructose in the cell?
Fructokinase
Which enzyme in fructose metabolism results in a product that can feed directly into glycolysis, linking the two pathways?
Aldolase B
Galactokinase
Phosphorylates galactose, trapping it in the cell
Galactose-1-phosphate uridyltransferase
Galactose –> glucose 1-phosphate + epimerase, linking glycolysis to galactose metabolism
Fructokinase
Works with hexokinase to phosphorylate fructose, trapping it in the cell
Aldolase B
Produces dihydroxyacetone phosphate (DHAP) and glyceraldehyde. Glyceraldehyde can be phosphorylated to glyceraldehyde 3-phosphate, linking glycolysis to fructose metabolism
What are the reactants for the pyruvate dehydrogenase (PDH) complex?
Pyruvate, NAD+ and CoA
What are the products for the pyruvate dehydrogenase (PDH) complex?
Acetyl-CoA, NADH and CO2
How does acetyl-CoA affect PDH complex activity?
Acetyl-CoA inhibits the PDH complex. As a product of the enzyme complex, a buildup of acetyl-CoA from either the citric acid cycle or fatty acid oxidation signals that the cell is energetically satisfied and that the production of acetyl-CoA should be slowed or stopped. Pyruvate can then be used to form other products, such as oxaloacetate for use in gluconeogenesis.
What is the structure of glycogen?
A core protein of glycogenin with linear chains of glucose emanating out from the center. Some of these chains are branches
What types of glycosidic links exist in a glycogen granule?
Alpha-1,4 glycosidic links (linear)
Alpha-1,6 glycosidic links (branched)
What are the two main enzymes of glycogenesis?
Glycogen synthase and branching enzyme
Glycogen synthase
Attaches the glucose molecule from UDP-glucose to the growing glycogen chain, forming an alpha-1,4 link in the process
Branching enzyme
Creates a branch by breaking an alpha-1,4 link in the growing chain and moving a block of oligoglucose to another location in the glycogen granule. The oligoglucose is then attached with an alpha-1,6 link
What are the two main enzymes of glycogenolysis?
Glycogen phosphorylase and debranching enzyme
Glycogen phosphorylase
Removes a glucose molecule from glycogen using a phosphate, breaking the alpha-1,4 link and creating glucose 1-phosphate
Debranching enzyme
Moves all of the glucose from a branch to a longer glycogen chain by breaking an alpha-1,4 link and forming a new alpha-1,4 link to the longer chain. The branch point is left behind; this is removed by breaking the alpha-1,6 link to form a free molecule of glucose
Under what physiological conditions should the body carry out gluconeogenesis?
When an individual has been fasting for >12 hours
To carry out gluconeogenesis, hepatic (and renal) cells must have enough energy to drive the process of glucose creation, which requires sufficient fat stops to under beta-oxidation
What are the four enzymes unique to gluconeogenesis?
Pyruvate carboxylase, phosphoenolpyruvate carboxylase (PEPCK), fructose-1,6-bisphosphatase, and glucose-6-phosphatase
Which enzyme does pyruvate carboxylase replace in glycolysis?
Pyruvate kinase
Which enzyme does phosphoenolpyruvate carboxylase (PEPCK) replace in glycolysis?
Pyruvate kinase
Which enzyme does fructose-1,6-bisphosphatase replace in glycolysis?
Phosphofructokinase-1 (PFK-1)
Which enzyme does glucose-6-phosphatase replace in glycolysis?
Glucokinase
How does acetyl-CoA shift the metabolism of pyruvate?
Acetyl-CoA inhibits PDH complex while activated pyruvate carboxylase. The net effect is to shift from burning pyruvate in the citric acid cycle to creating new glucose molecules for the rest of the body. The acetyl-CoA for this regulation comes predominantly from beta-oxidation, not glycolysis.
What are the two major metabolic products of the pentose phosphate pathway (PPP)?
Ribulose 5-phosphate and NADPH
What are three primary functions of NADPH?
Involved in lipid biosynthesis
Bacterial bleach formation uncertain while blood cells
Maintenance of glutathione stores to protect against reactive oxygen species
GLUT 2
Found in the liver (for glucose storage) and pancreatic beta-islet cells (as part of the glucose sensor)
Has a high Km value
GLUT 4
Found in adipose tissue and muscle
Stimulated by insulin
Has a low Km value
Where does glycolysis occur?
In the cytoplasm
Does glycolysis require oxygen?
No
How many ATP molecules are released per glucose after glycolysis is completed?
2
Important glycolytic enzymes
Glucokinase Hexokinase Phosphodructokinase-1 (PFK-1) Phosphofructokinase-2 (PFK-2) Glyceraldehyde-3-phosphate dehydrogenase 3-phosphoglycerate kinase Pyruvate kinase
Where is glucokinase?
Pancreatic beta-islet cells (part of the glucose sensor) Liver cells (responds to insulin)
Where is hexokinase?
Peripheral tissues
What is the rate limiting step of glycolysis?
PFK-1: fructose 6-phosphate –> fructose 1,6-bisphosphate
What is PFK-1 activated by?
AMP and fructose 2,6-bisphosphate (F2,6-BP)
What is PFK-1 inhibited by?
ATP and citrate
Phosphofructokinase-2 (PFK-2)
Produces F2,6-BP, which activates PFK-1
What is PFK-2 activated by?
Insulin
What is PFK-2 inhibited by?
Glucagon
Which steps perform substrate-level phosphorylation in glycolysis?
3-phosphoglycerate kinase and pyruvate kinase
Substrate-level phosphorylation
Making ATP by placing an inorganic phosphate (Pi) onto ADP
Which steps are irreversible in glycolysis?
Glucokinase/hexokinase
PFK-1
Pyruvate kinase
What happens to the NADH produced by glycolysis when oxygen and mitochondria are present?
It is oxidized by the mitochondrial electron transport chain
What happens to the NADH produced by glycolysis when oxygen and mitochondria are absent?
It is oxidized by cytoplasmic lactate dehydrogenase
e.g. RBCs, skeletal muscles (during short, intense Burts of exercise) and any cell deprived of oxygen
How do we acquire galactose?
From lactose in milk
What traps galactose in the cell?
Galactokinase
How do we acquire fructose?
From honey, fruit and sucrose (common table sugar)
What traps fructose in the cell?
Fructokinase
What stimulates the PDH complex?
Insulin
What inhibits the PDH complex?
Acetyl-CoA
Glycogenesis
Glycogen synthesis
What activates glycogen synthase in the liver and muscles?
Insulin
Glycogenolysis
The breakdown of glycogen
What activates glycogen phosphorylase in the liver?
Glucagon
What activates glycogen phosphorylase in the exercising skeletal muscles?
Epinephrine and AMP
Where does gluconeogenesis occur?
In the liver and kidney cells’ cytoplasm and mitochondria
Does gluconeogenesis occur more in the liver or the kidneys?
Liver
Gluconeogenesis
The reverse of glycolysis, using the same enzymes except pyruvate kinase, PFK-1 and glucokinase
Pyruvate carboxylase
Pyruvate –> oxaloacetate
Phosphoenolpyruvate carboxykinase (PEPCK)
Oxaloacetate –> phosphoenolpyruvate
What activates pyruvate carboxylase?
Acetyl-CoA (from beta-oxidation)
What activates PEPCK?
Glucagon and cortisol
Fructose-1,6-bisphosphatase
Fructose 1,6-bisphosphate –> fructose 6-phosphate
What is the rate limiting step of gluconeogenesis?
Fructose-1,6-bisphosphatase: fructose 1,6-bisphosphate –> fructose 6-phosphate
What directly activates fructose-1,6-bisphosphatase?
ATP
What indirectly activates fructose-1,6-bisphosphatase?
Glucagon (via decreased levels of fructose 2,6-bisphosphate)
What directly inhibits fructose-1,6-bisphosphatase?
AMP
What indirectly inhibits fructose-1,6-bisphosphatase?
Insulin (via increased levels of fructose 2,6-bisphosphate)
Glucose-6-phosphatase
Glucose 6-phosphate –> glucose
Where is glucose-6-phosphatase?
Endoplasmic reticulum of liver cells
Where does pentose phosphate pathway (PPP) (hexose monophosphate [HMP] shunt) occur?
Cytoplasm
What is the rate limiting step of PPP?
Glucose-6-phosphate dehydrogenase
What activates glucose-6-phosphate dehydrogenase?
NADP+ and insulin
What inhibits glucose-6-phosphate dehydrogenase?
NADPH
A man collapses while running a marathon and is taken to the ER. His blood is found to be somewhat acidic, and further tests show increased lactate dehydrogenase activity. This enzyme is involved in which pathway?
Anaerobic glycolysis in muscles
Does the liver require a constant supply of glucose from the blood for energy during a fast?
No because it can produce its own glucose through gluconeogenesis
Does the kidney require a constant supply of glucose from the blood for energy during a fast?
No because it can produce its own glucose through gluconeogenesis
When fatty acid beta-oxidation predominates in the liver, mitochondrial pyruvate is most likely to be:
Carboxylated to oxaloacetate for entry into gluconeogenesis
A biopsy is done on a child with an enlarged liver and shows accumulation of glycogen granules with single glucose residues remaining a the branch points near the periphery of the granule. The most likely genetic defect is in the gene encoding:
Debranching enzyme complex
After a brief period of intense exercise, the activity of muscle pyruvate dehydrogenase is greatly increased. This increased activity is most likely due to:
Increased pyruvate concentration
After a large meal, would glucagon be in high concentration?
No, because it is used to raise blood sugar. After eating, blood sugar is already high.
A man is given antibiotics to treat a UTI and develops an episode of RBC lysis. Further studies show weakness of the plasma membrane and Heinz bodies (collections of oxidized hemoglobin). What is the most likely defective enzyme in this patient?
Glucose-6-phosphate dehydrogenase
PPP’s responsibility is generating NADPH. In individuals with G6PD deficiency, NADPH cannot be produced at sufficient levels and oxidative stresses lead to cell membrane and protein (hemoglobin) damage
What is the overall reaction of the pyruvate dehydrogenase complex?
Pyruvate + CoA-SH + NAD+ –> acetyl-CoA + CO2 + NADH + H+
What molecules can be used to make acetyl-CoA?
Fatty acids, ketogenic amino acids, ketones, and alcohol
How does the body convert fatty acids to acetyl-CoA?
Shuttle acyl group from cytosolic CoA-SH to mitochondrial CoA-SH via carnitine –> undergo beta-oxidation
Fatty acid couples with CoA in the cytosol –> fatty acyl-CoA –(moves into the intermembrane space)–> the fatty acid group (acyl group) is transferred to carnitine –> acyl-carnitine –(crosses the inner membrane)–> the acyl group is transferred to a mitochondrial CoA –> fatty acyl-CoA reforms –(undergoes beta oxidation)–> acetyl-CoA
How does the body convert ketogenic amino acids to acetyl-CoA?
Transaminate to lose nitrogen –> convert carbon skeleton into ketone body –> ketone body is converted into acetyl-CoA
How does the body convert ketones to acetyl-CoA?
Reverse of ketone body formation
How does the body convert alcohol to acetyl-CoA?
Alcohol dehydrogenase and acetaldehyde dehydrogenase convert alcohol into acetyl-CoA
What is the purpose of all the reactions that collectively make up the citric acid cycle?
Complete oxidation of carbons in intermediates to CO2 so that reduction reactions can be coupled with CO2 formation, thus forming energy carriers such as NADH and FADH2 for the electron transport chain
What enzyme catalyzes the rate-limiting step of the citric acid cycle?
Isocitrate dehydrogenase
What are the three main sites of regulation within the citric acid cycle?
Citrate synthase
Isocitrate dehydrogenase
Alpha-ketoglutarate complex
What molecule(s) inhibit(s) citrate synthase?
ATP
NADH
Succinyl-CoA
Citrate
What molecule(s) inhibit(s) isocitrate dehydrogenase?
ATP
NADH
What molecule(s) inhibit(s) citrate alpha-ketoglutarate complex?
ATP
NADH
Succinyl-CoA
What molecule(s) activate(s) citrate synthase?
Nothing
What molecule(s) activate(s) isocitrate dehydrogenase?
ADP
NAD+
What molecule(s) activate(s) alpha-ketoglutarate complex?
ADP
Ca^2+
Which complex in ETC is associated with pumping a proton into the intermembrane space?
Complex I, III and IV
Which complex in ETC is associated with acquiring electrons from NADH?
Complex I
Which complex in ETC is associated with acquiring electrons from FADH2?
Complex II
Which complex in ETC is associated with having the highest reduction potential?
Complex IV
What role does the electron transport chain play in the generation of ATP?
The electron transport chain generates the proton-motive force, an electrochemical gradient across the inner mitochondrial membrane, which provides the energy for ATP synthase to function
Based on its needs, which of the two shuttle mechanisms in ETC is cardiac muscle most likely to utilize?
The malate-aspartate shuttle
This mechanism is the more efficient one, it makes sense for a highly aerobic organ such as the heart to utilize it in order to maximize its ATP yield
What is the difference between the ETC and oxidative phosphorylation?
The ETC is made up of the physical set of inter membrane proteins located on the inner mitochondrial matrix, and they undergo oxidation-reduction reactions as they transfer electrons to oxygen, the final electron acceptor. As electrons are transferred, a proton-motive force is generated in the intermembrane space.
Oxidative phosphorylation is the process by which ATP is generated via harnessing the proton gradient, and it utilizes ATP synthase to do so.
What links ETC and oxidative phosphorylation?
As electrons are transferred to oxygen in ETC, a proton-motive force is generated in the inter membrane space and oxidative phosphorylation uses that proton gradient to make ATP
The delta G of NADH reducing oxygen directly is significantly greater than any individual step along the electron transport chain. If this is the case, why does transferring electrons along the ETC generate more ATP than direct reduction of oxygen by NADH?
By splitting up electron transfer into several complexes, enough energy is released to facilitate the creation of a proton gradient at many locations, rather than just one. The greater the proton gradient is, the greater the ATP generation will be. Direct reduction of oxygen by NADH would release a significant amount of energy to the environment, resulting in inefficient electron transport.
Acetyl-CoA
Contains a high-energy thirster bond that can be used to drive other reactions when hydrolysis occurs
Pyruvate dehydrogenase complex
A five-enzyme complex in the mitochondrial matrix that forms, and is also inhibited by, acetyl-CoA and NADH
Enzymes in the pyruvate dehydrogenase complex
Pyruvate dehydrogenase (PDH) Dihydrolipoyl transacetylase Dihydrolipoyl dehydrogenase Pyruvate dehydrogenase kinase Pyruvate dehydrogenase phosphatase
Pyruvate dehydrogenase (PDH)
Pyruvate –(oxidation)–> CO2 + 2 carbon molecule
Requires thiamine pyrophosphate (vitamin B1, TPP) and Mg^2+
Dihydrolipoyl transacetylase
2 carbon molecule –(oxidation)–> acetyl group
Transfers acetyl group to CoA –> acetyl-CoA
Uses lipoic acid
Dihydrolipoyl dehydrogenase
Reoxidizes lipoic acid –> FADH2 (which can later transfer electrons to NAD+ –> NADH, which can then feed into ETC)
Uses FAD
Pyruvate dehydrogenase kinase
PDH –(phosphorylation)–> PDH turned off
Activated when ATP or Acetyl-CoA are present
Pyruvate dehydrogenase phosphatase
PDH –(dephosphorylation)–> PDH turned on
Activated when ADP levels are high
Where does the citric acid cycle take place?
The mitochondrial matrix
What is the main purpose of the citric acid cycle?
Oxidize carbon in intermediates to CO2 and generate high-energy electron carries (NADH and FADH2) and GTP
What are the enzymes in the citric acid cycle?
Citrate synthase Aconitase Isocitrate dehydrogenase Alpha-ketoglutarate dehydrogenase complex Succinyl-CoA synthetase Succinate dehydrogenase Fumarase Malate dehydrogenase
Citrate synthase
Couples acetyl-CoA to oxaloacetate –(hydrolysis)–> citrate + CoA-SH
How is citrate synthase regulated?
Negative feedback from ATP, NADH, succinyl-CoA and citrate
Aconitase
Citrate –(isomerize)–> isocitrate
Isocitrate dehydrogenase
Isocitrate –(oxidation and decarboxylation)–> alpha-ketoglutarate
Generates the first CO2 and first NADH of the cycle
The rate-limiting step of the citric acid cycle
How is isocitrate dehydrogenase regulated?
Inhibitors: ATP and NADH
Activators: ADP and NAD+
Alpha-ketoglutarate dehydrogenase complex
Alpha-ketoglutarate –(metabolism)–> succinyl-CoA
Acts like the PDH complex
Generates the second CO2 and second NADH of the cycle
How is alpha-ketoglutarate dehydrogenase complex regulated?
Inhibition: ATP, NADH and succinyl-CoA
Activation: ADP and Ca^2+
Succinyl-CoA synthetase
Succinyl-CoA –(hydrolysis of thirster bond)–> succinate + CoA-SH
Generates the one GTP generated in the citric acid cycle
Succinate dehydrogenase
Succinate –(succinate dehydrogenase)–> fumarate
A flavoprotein that is anchored in the inner mitochondrial membrane because it requires FAD, which is reduced to form the one FADH2 generated in the citric acid cycle
Fumarase
Fumarate –(hydrolysis of the alkene bond)–> malate
Malate dehydrogenase
Malate –(oxidation)–> oxaloacetate
Generates the third and final NADH of the cycle
Where does the electron transport chain take place?
On the matrix-facing surface of the inner mitochondrial membrane
Electron transport chain
NADH donates electrons to the chain, which are passed from one complex to the next
As ETC progresses, reduction potentials increase until oxygen, which has the highest reduction potential, receives the electrons
Complexes in ETC
I (NADH-CoQ oxidoreductase)
II (Succinate-CoQ oxidoreductase)
III (CoQH2-cytochrome c oxidoreductase)
IV (cytochrome c oxidase)
Complex I (NADH-CoQ oxidoreductase)
Transfers electrons from NADH to flavin mononucleotide (FMN) –> transfers electrons from FMN to coenzyme Q (CoQ) –> CoQH2 forms
4 H+ are translocated
Uses an iron-sulfur cluster
Complex II (Succinate-CoQ oxidoreductase)
Transfers electrons from succinate to FAD –> transfers electrons from FAD to CoQ –> CoQH2 forms
No H+ are translocated
Uses an iron-sulfur cluster
Complex III (CoQH2-cytochrome c oxidoreductase)
Transfers electrons from CoQH2 to heme –> cytochrome c forms as part of the Q cycle
4 H+ are translocated
Uses an iron-surfer cluster
Complex IV (cytochrome c oxidase)
Transfers electrons in the form of hydride ions (H-) from cytochrome c to oxygen –> water forms
2 H+ are translocated
Uses Cu^2+
Can NADH cross the inner mitochondrial membrane?
No
How does NADH cross the inner mitochondrial membrane?
Using glycerol 3-phosphate shuttle or malate-aspartate shuttle
Glycerol 3-phosphate shuttle
Electrons are transferred from NADH to dihydroxyacetone phosphate (DHAP) –> glycerol 3-phosphate forms
Electrons then move to mitochondrial FAD –> FADH2 forms
Malate-aspartate shuttle
Electrons are transferring from NADH to oxaloacetate –> malate forms –> Malate crosses the inner mitochondrial membrane –> Malate transfers electrons to mitochondrial NAD+ –> NADH forms
Proton-motive force
The electrochemical gradient generated by the electron transport chain across the inner mitochondrial membrane
Which has a higher concentration of protons: the intermembrane space or the matrix?
The innermembrane space
This gradient stores energy, which can be used to form ATP via chemiosmotic coupling
ATP synthase
The enzyme responsible for generating ATP from ADP and inorganic phosphate (Pi)
Has an F0 portion and an F1 portion
F0 portion of ATP synthase
AN ion channel
Allows protons to flow down the gradient from the intermembrane space to the matrix
F1 portion of ATP synthase
Uses the energy released by the gradient to phosphorylate ADP into ATP
Energy yield from glycolysis
2 NADH
2 ATP
Energy yield from PDH
1 NADH/molecule of pyruvate
2NADH/molecule of glucose
Energy yield from TCA
3 NADH + 1 FADH2 + 1 GTP/pyruvate molecule
6 NADH + 2 FADH2 + 2 GTP/ glucose molecule
Energy yield from NADH
2.5 ATP
How much NADH is produced from 1 glucose molecule by the end of TCA?
10
What is the energy yield from 1 molecule of glucose by the end of TCA?
25 ATP (from total NADH) + 3 ATP (from total FADH2) + 2 ATP (from total GTP) + 2 ATP (from glycolysis) = 32 ATP
How much FADH2 is produced from 1 glucose molecule by the end of TCA?
2
Energy yield from FADH2
1.5 ATP
Energy yield from GTP
1 ATP
How much GTP is produced from 1 glucose molecule by the end of TCA?
2
Fatty acids enter the catabolic pathway in the form of:
Acetyl-CoA
In which part of the cell us cytochrome c located?
Inner mitochondrial membrane
Why is it preferable to cleave thioester links rather than typical ester links in aerobic metabolism?
Thioester hydrolysis has a higher energy yield
Why can cytosolic NADH potentially yield less ATP than mitochondrial NADH?
Because electron transfer from cytosol to matrix can take more than one pathway
In high doses, aspirin functions as a mitochondrial uncoupler. How would this affect glycogen stores?
It causes depletion of glycogen stores
Uncouplers inhibit ATP synthesis without affecting the electron transport chain. Because the body must burn more fuel to maintain the proton-motive force, glycogen stores will be mobilized to feed into glycolysis, then the TCA and finally oxidative phosphorylation.
Which complex does not contribute to the proton motive force?
Complex II
What directly provides the energy needed to form ATP in the mitochondrion?
An electrochemical proton gradient
When lipids leave the stomach, what stages of digestion have been accomplished?
Physical digestion: accomplished in the mouth and stomach, reducing the particle size
When lipids leave the stomach, what enzymes are added to accomplish the next phase?
Chemical digestion: small intestine, pancreatic lipase, collapse, cholesterol esterase and bile
Absorption occurs in a more distal portion of the small intestine
Do all lipids enter the circulation through the lymphatic system?
No, small free fatty acids enter the circulation directly
What conditions and hormones promote lipid mobilization from fat stores?
In the post absorptive and prolonged fasting states, lipid mobilization is favored. A decrease in insulin levels, as well as an increase in epinephrine or cortisol, will increase lipid mobilization from adipocytes.
What is the ratio of free fatty acids to glycerol produced through lipid mobilization?
3:1 (a triacylglycerol molecule is composed of glycerol and three fatty acids)
What is the primary method of transporting free fatty acids in the blood?
Free fatty acids remain in the blood, bonded to albumin and other carrier proteins
A smaller amount will remain unbounded
Order the lipoproteins from greatest percentage of protein to least percentage of protein. Which one is primarily involved in triacylglycerol transport?
- HDL
- LDL
- IDL
- VLDL
- chylomicrons
VLDL and chylomicrons are the primary triacylglycerol transporters
HDL and LDL are mostly involved in cholesterol transport
Lipoproteins are synthesized primarily by which two organs?
Intestine and liver
Under what conditions is HMG-CoA reductase most active?
The absence of cholesterol
Stimulated by insulin
In what cellular region does HMG-CoA reductase exist?
smooth endoplasmic reticulum
What proteins are specific to the formation and transmission of cholesteryl esters and what are their functions?
LCAT catalyzes the esterification of cholesterol to form cholesteryl esters
CETP promotes the transfer of cholesterol esters from HDL to IDL, forming LDL
What are the five steps in the addition of acetyl-CoA to a growing fatty acid chain?
- Attachment to acyl carrier protein
- Bond formation between molecules
- Reduction of carbonyl group
- Dehydration
- Reduction of double bond
How does beta-oxidation of unsaturated fatty acids differ from that of saturated fatty acids?
There is an additional isomerase and an additional reductase for the beta-oxidation of unsaturated fatty acids, which provide the stereochemistry necessary for further oxidation
Are fatty acids synthesized in the cytoplasm and modified by enzymes in the smooth endoplasmic reticulum?
Yes
Why are fatty acids used to create ketone bodies instead of creating glucose?
Fatty acid degradation results in large amounts of acetyl-CoA, which cannot enter the gluconeogenic pathway to produce glucose. Only odd-numbered fatty acids can act as a source of carbon for gluconeogenesis; even then, only the final malonyl-CoA molecule can be used. Energy is packaged into ketone bodies for consumption by the brain and muscles
What conditions and tissues favor ketogenesis?
Prolonged fast
Occurs in the liver
Stimulated by increasing concentrations of acetyl-CoA
What conditions and tissues favor ketolysis?
Prolonged fast
Stimulated by a low-energy state in muscle and brain tissues
Does not occur in the liver
Are bodily proteins commonly broken down to provide acetyl-CoA for lipid synthesis?
No
Proteins are more valuable to the cell than lipids, thus they will not commonly be broken down for lipid synthesis
Where does the bulk of protein digestion occur?
Small intestine
During protein processing, what is the eventual fate of the carbon skeleton?
Transported to the liver for processing into glucose or ketone bodies
During protein processing, what is the eventual fate of the amino group?
Will feed into the urea cycle for excretion
During protein processing, what is the eventual fate of the side chains?
Processed depending on their composition
Basic chains: will be processed like amino acids (will feed into the urea cycle for excretion)
Other functional groups: will be treated like the carbon skeleton (transported to the liver for processing into glucose or ketone bodies)
Where does the mechanical digestion of lipids occur?
Mouth and stomach
Where does the chemical digestion of lipids occur?
Small intestine
Which enzymes in the small intestine are used to chemically digest lipids?
Bile, pancreatic lipase, collapse and cholesterol esterase
What happens to digested lipids?
They can either form micelles for absorption or be absorbed directly
How are short-chain fatty acids absorbed?
Across the intestine into the blood
How are long-chain fatty acids absorbed?
As micelles and assembled into chylomicrons for release into the lymphatic system
Lipids are mobilized from adipocytes by
Hormone-sensitive lipase
Lipids are mobilized from lipoproteins by
Lipoprotein lipase
Chylomicrons
Transport mechanism for dietary triacylglycerol molecules and are transported via the lymphatic system
VLDL
Transports newly synthesized triacylglycerol molecules from the liver to peripheral tissues in the bloodstream
IDL
VLDL remnant in transition between triacylglycerol and cholesterol transport
Picked up cholesteryl esters from HDL
LDL
Primarily transports cholesterol for use by tissues
HDL
Involved in the reverse transport of cholesterol
Apoproteins
Control interactions between lipoproteins
How can cholesterol be obtained?
Dietary sources
de novo synthesis in the liver
De novo
Synthesis of a complex molecule, which refers to the biochemical pathway where a complex biomolecule is synthesized anew from simple molecules
What is the key enzyme in cholesterol biosynthesis?
HMG-CoA reductase
LCAT
Catalyzes the formation of cholesteryl esters for transport with HDL
CETP
Catalyzes the transient of IDL to LDL by transferring cholesteryl esters from HDL
Fatty acids
Carboxylic acids, typically with a single long chain, although they can be branched
Saturated fatty acids
Have no double bonds between carbons
Unsaturated fatty acids
Have one or more double bonds
Fatty acids synthesis
Location: cytoplasm (from acetyl-CoA) and transported out of the mitochondria
Steps: activation, bond formation, reduction, dehydration and another reduction (repeated 8 times to form palmitic acid)
What is the only fatty acid that humans can synthesize?
Palmitic acid
Fatty acid oxidation
Location: mitochondria (following transport by the carnitine shuttle)
Steps: beta-oxidation uses cycles of oxidation, hydration, oxidation and cleavage (branched and unsaturated fatty acids require special enzymes – unsaturated fatty acids use isomerase and an additional reductase during cleavage)
Ketone bodies
Form ketogenesis during a prolonged starvation state due to excess acetyl-CoA in the liver
Ketolysis
Regenrated acetyl-CoA for use as an energy source in peripheral tissues
How much energy can the brain derive from ketone bodies during prolonged starvation?
2/3 of its original energy capacity
Where does protein digestion occur?
Small intestine
When does catabolism of cellular proteins occur?
Under conditions of starvation
What is the carbon skeleton of amino used for after protein catabolism?
Energy (through gluconeogenesis or ketone body formation)
What is the amino groups of amino used for after protein catabolism?
Urea cycle (for excretion)
During fatty acid mobilization, what happens?
HSL is activated and free fatty acids are released
During fatty acid mobilization, there is a breakdown of triacylglycerols in adipocytes by hormone-sensitive lipase (HSL). This breakdown results in the release of three fatty acids and a glycerol molecule. The glycerol may be used by the liver for gluconeogenesis, but adipocytes do not have the ability to carry out gluconeogenesis.
How do chylomicrons and VLDL differ?
Chylomicrons are synthesized in the instant and VLDL are synthesized in the liver
What happens in the absence of apolipoproteins?
- An inability to secrete lipid transport lipoproteins
- An inability to endocytose lipoproteins
- A decreased ability to remove excess cholesterol from blood vessels
While the transport and lipid binding functions of most lipoproteins are independent of apolipoprotein component, the interaction of these lipoproteins with the environment is controlled almost exclusively by apolipoproteins. Lipoproteins cannot exit or enter cells without apolipoproteins and are unable to transfer lipids without specialized apolipoproteins or cholesterol-specific enzymes.
Statin drugs inhibit HMG-CoA reductase. As such, they are likely prescribed for
High cholesterol
Palmitic acid short hand notation
16:0 (16 carbons + no double bonds)
Where does beta-oxidation occur?
Mitochondria
The majority of triacylglycerols stored in adipocytes originate from:
Synthesis in the liver
2,4-dienoyl-CoA reductase is used in the oxidation of:
Polyunsaturated fatty acids
What conditions does delta G’ adjust for that are not considered with delta G?
pH (it fixes it at 7)
Other conditions (temperature and concentration of reagents) are still fixed at their values from standard conditions and must be adjusted for if they are not 1 M
Why can heat be used as a measure of internal energy in living systems?
The cellular environment has a relatively fixed volume and pressure, which eliminates work from out calculations of internal energy
If delta U = Q - W and W = 0, then delta U = Q
When delta H is positive and entropy is positive, is the reaction spontaneous?
Yes, but only in high temperatures
When delta H is positive and entropy is negative, is the reaction spontaneous?
No
When delta H is negative and entropy is positive, is the reaction spontaneous?
Yes
When delta H is negative and entropy is negative, is the reaction spontaneous?
Yes, but only in low temperatures
How does coupling with ATP hydrolysis alter the energetics of a reaction?
ATP hydrolysis yields about 30 kJ/mol of energy, which can be harnessed to drive other reactions forward. This may either allow a non spontaneous reaction to occur or increase the rate of a spontaneous reaction.
Explain why ATP is an inefficient molecule for long-term energy storage.
ATP is an intermediate-energy storage molecule and is not energetically dense. The high-energy bonds in ATP and the presence of a significant charge make it an inefficient molecule to pack into a spall space. Long-erm storage molecules are characterized by energy density and stable, non repulsive bonds, primarily seen in lipids.
What is an advantage of analyzing the half-reactions in biological oxidation and reduction reactions?
Analyzing half-reactions can help to determine the number of electrons being transferred. This type of analysis also facilitates balancing equations and the determination of electrochemical potential if reduction potentials are provided.
Soluble electron carriers
NADH, NADPH, Ubiquinone (CoQ), Cytochromes, Glutathione
Which metabolic pathways use NADH?
Glycolysis, fermentation, citric acid cycle, electron transport chain
Which metabolic pathways use NADPH?
Pentose phosphate pathway, lipid biosynthesis, bleach formation, oxidative stress and photosynthesis
Which metabolic pathways use ubiquinone (CoQ)?
Electron transport chain
Which metabolic pathways use cytochromes?
Electron transport chain
Which metabolic pathways use glutathione?
Oxidative stress
Provide an example of disequilibrium that is maintained at the expense of cellular energy?
Any excitable cells is maintained in a state of disequilibrium. Classic examples include muscle tissue and neurons. In addition, cell volume and membrane transport are regulated by the action of the sodium-potassium pump, which can maintain a stable disequilibrium state in most tissues.
What tissue is least able to change its fuel source in periods of prolonged starvation?
RBCs
Cells that rely solely on anaerobic respiration are the least adaptable to different energy sources. Therefore, red blood cells are the least flexible during periods of prolonged starvation and stay reliant on glucose.
During what stage is there the greatest decrease in the circulating concentration of insulin?
During the post absorptive state, there is the greatest decrease in insulin levels. The concentrations of the counter regulatory hormones (glucagon, cortisol, epinephrine, norepinephrine and growth hormone) begin to rise.
What is the primary metabolic function of insulin?
Promotes glucose uptake by adipose tissue and muscles, glucose utilization in muscle cells, and macromolecule storage (glycogenesis and lipogenesis)
What is the primary metabolic function of glucagon?
Increases blood glucose levels by promoting glycogenolysis, gluconeogenesis, lipolysis and ketogenesis
What is the primary metabolic function of cortisol?
Increases lipolysis and amino acid mobilization, decreases glucose uptake in certain tissues and engines the activity of counterrgulatory hormones
What is the primary metabolic function of catecholamines?
Increase glycogenolysis in muscle and liver cells and increases lipolysis in adipose tissue
What is the primary metabolic function of thyroid hormones (T3/T4)?
Increase basic metabolic rate and potentiate the activity of other hormones
Thyroid storm is a potentially lethal state of extreme hyperthyroidism in which T3 and T4 levels are significantly above normal limits. What vital sign abnormalities might be expected in a patient with thyroid storm?
Hyperthermia, tachycardia, hypertension and tachypnea
What is the preferred fuel for most cells in the well-fed state?
Glucose (except cardiac muscles prefer fatty acids)
What organ consumes the greatest amount of glucose relative to its percentage of body mass?
Brain
Describe the major metabolic functions of the liver
Maintaining a steady-state concentration go glucose in the blood through glucose uptake and storage, glycogenolysis and gluconeogenesis
Participates in cholesterol and fat metabolism, the urea cycle, bile synthesis, and the detoxification of foreign substances
How is the respiratory quotient expected to change when a person transitions from resting to brief exercise?
As a person begins to exercise, the proportion of energy derived from glucose increases. This transition to almost exclusively carbohydrate metabolism will cause the respiratory quotient to approach 1.
Can body mass be predicted by the leptin receptor phenotype and caloric intake alone?
No: energy expenditure, genetics, socioeconomic status, geography and other hormones also play a role in body mass regulation
Is it easier to gain weight than to lose it?
Yes: the threshold is lower for uncompensated weight gain that it is for uncompensated weight loss. Therefore, it is easier to surpass this threshold and gain weight that to lose it.
Gibbs free energy (delta G)
= enthalpy - (temperature x entropy)
= delta H - T delta S
Modified standard state (delta G)
= delta G + R T ln (Q)
Respiratory quotient
= CO2 produced / O2 consumed
Estimates the composition of fuel that is actively consumed by the body
Body mass index (BMI)
= mass / height^2
Changes in enthalpy in a closed biological system is equal to:
Changes in internal energy, which is equal to heat exchange within the environment
Can work be performed in a closed system?
No, because pressure and volume remain constant
Entropy
A measure of energy dispersion in a system
ATP
A mid-level energy molecule
Contains high-energy phosphate bonds that are stabilized upon hydrolysis by resonance, ionization and loss of charge repulsion
Provides energy through hydrolysis and coupling to energetically unfavorable reactions
Can participate in phosphorylation group transfers as a phosphate donor
Half-reactions
Provide useful information about stoichiometry and thermodynamics
Flavoproteins
One subclass of electron carriers that are derived from riboflavin (vitamin B2)
Riboflavin
Vitamin B2
Is equilibrium a desirable state for biochemical reactions?
No because organisms need to harness free energy to survive
Postprandial/well-fed (absorptive) state
Insulin secretion is high and anabolic metabolism prevails
Postabsorptive (fasting) state
Insulin section decreases while glucagon and catecholamine secretion increases
Observed in short-term fasting
There is a transition to catabolic metabolism
Prolonged fasting (starvation) state
Dramatically increases glucagon and catecholamine section
Most tissues rely on fatty acids
At maximum, 2/3 of the brain’s energy can be derived from ketone bodies
Insulin
Decreases blood glucose by increasing cellular uptake
Increases the rate of anabolic metabolism
Secreted by pancreatic beta-cells
Regulated by blood glucose levels
Glucagon
Increases blood glucose by promoting gluconeogenesis and glycogenolysis in the liver
Secreted by pancreatic alpha-cells
Stimulated by low glucose and high amino acid levels
Glucocorticoids
Increase blood glucose in response to stress by mobilizing fat stores and inhibiting glucose uptake
Increase the impact of glucagon and catecholamines
Catecholamines
Promote glycogenolysis and increase basal metabolic rate through their sympathetic nervous system activity
Thyroid hormones
Modulate the impact of other metabolic hormones and have a direct impact on basal metabolic rate
Which is more potent: T3 or T4?
T3
Which has a shorter half-life: T3 or T4?
T3
Which is present in low concentrations in the blood: T3 or T4?
T3
What happens to T4 in tissues?
It gets converted to T3
What is the most metabolically diverse organ?
Liver
Hepatocytes
Responsible for the maintenance of blood glucose levels by glycogenolysis and gluconeogenesis in response to pancreatic hormone stimulation
Participate in the processing of lipids and cholesterol, bile, urea and toxins
Adipose tissue
Stores lipids under the influence of insulin and releases them under the influence of epinephrine
Skeletal muscle metabolism in resting muscles
Conservation of carbohydrates in glycogen stores and use of free fatty acids from the bloodstream
Skeletal muscle metabolism in active muscles
Anaerobic metabolism, oxidative phosphorylation (using glucose), direct phosphorylation (using creatine phosphate), or fatty acid oxidation
Depends on the fiber type and exercise duration
Cardiac muscle
Uses fatty acid oxidation in both the well-fed and fasting states
Brain and other nervous tissue
Consume glucose in all metabolic states, except for prolonged fasts, where up to 2/3 of the brain’s fuel may come from ketone bodies
How can metabolic rates be measures?
Calorimetry, respirometry, consumption tracking and measurement of blood concentrations of substrates and hormones
Hormones that play a role in body mass
Leptin, ghrelin and orexin
Is the free energy of ATP hydrolysis independent of pH?
No because a protonated ATP molecule contains less negative charge and therefore experiences less repulsive force
Is the free energy of ATP hydrolysis nearly the same as for ADP hydrolysis?
Yes
How do hormonal controls of glycogen metabolism differ from allosteric controls?
Hormonal controls are coordinated to regulate the metabolic activity of the entire organism, while allosteric controls can be local or systemic.
Therefore, the hormonal control of glycogen is only systemic.
The modification of the enzymes of glycogen metabolism by insulin and glucagon is either through phosphorylation or dephosphorylation, both of which modify covalent bonds.
Therefore, the hormonal control of glycogen is systemic and covalent.
Which is more dependent on insulin: active or resting skeletal muscles?
Resting skeletal muscles because (like adipose tissue) they require insulin for glucose uptake. Active skeletal muscles use creatine phosphate and glycogen (regulated by epinephrine and AMP) to maintain its energy requirements.
Glucocorticoids have been implicated in stress-related weight gain because:
They increase glucose levels, causes insulin secretion
Respiratory quotient (RQ) = 0.7
The body is primarily metabolizing lipids
Respiratory quotient (RQ) = 0.8 - 0.9
The body is primarily metabolizing amino acids
Respiratory quotient (RQ) = 1
The body is primarily metabolizing carbohydrates
Leptin
Decreases appetite by inhibiting the production of Orexin
Orexin
Associated with alertness
How many times are ATP stores turned over daily?
1000 times per day
Prolonged fast stages
- Increase in glucagon, which accomplishes its cellular activity by phosphorylating and dephosphorylating metabolic enzymes
- Glycogen storage is halted
- Protein breakdown
- Ketone bodies are used by the brain as its main energy source
