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Biochemistry-Erin > Biochem Exam I > Flashcards

Biochem Exam I Flashcards

1
Q

What is sequence specific conformation?

A

some amino acids tend to favor one structure over the other due to chemical or physical properties
ex. glutamine prefers alpha helix bc there is less of a steric hindrance when forming

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2
Q

What did Christian Arfisen discover about protein folding?

A

-he experimented with many proteins, putting them in relaxed states (breaking bonds) and then slowly placing them back into the conditions from which they came to determine how the protein folded itself. He also discovered scrambled proteins (incorrect folding).

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3
Q

What are the 4 determinants of protein folding?

A
  • secondary structure allows for efficient packing
  • folding is hierarchical
  • context dependent
  • hydrophobic effect
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4
Q

How do you calculate an amino acid’s propensity for a secondary structure?

A

f alpha= aa in alpha helix/all aa

P= f alpha/f (alpha)[avg aa]

P= propensity/natural inclination

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5
Q

What is a folding funnel?

A

This is a graph that theoretically determines what shape a protein will take based on entropy and energy of the conformation. A protein will be most stable with low entropy and energy levels, this will be the native state.

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6
Q

What is a molten globule state?

A
  • an intermediate conformational state between the native and the fully unfolded states of a globular protein.
  • a compact globule with a “molten” side chain structure that is primarily stabilized by nonspecific hydrophobic interactions
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7
Q

What are the characteristics of a molten globule state?

A
  • presence of a native-like content of secondary nature
  • the absence of a specific tertiary structure produced by the tight packing of aa chains
  • compactness in the overall shape of the protein molecule, with a radius 10-30% larger than that of the native state
  • presence of a loosely packed hydrophobic core that increases the hydrophobic surface area accessible to solvent
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8
Q

Alternate conformations

A
  • different conformations are possible with the same polypeptide chain based on environmental factors (pH, temp, etc) and the need for a specific conformation/protein
  • example: lymphotactin
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9
Q

What are IUPs?

A

Intrinsically Unstructured Proteins

  • can adopt 2 different structures only one of which results in protein aggregation and pathological conditions
  • no discreet 3D structure
  • -unstructured regions are rich in charged and polar aa with a few hydrophobic residues
  • important in signaling and regulatory pathway
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10
Q

What interactions govern protein folding?

A

-non-covalent interactions
+short range repulsion(much like a magnet, must have low energy to interact)
+ electrostatic forces
+van der waals interactions
+hydrogen bonds (alpha helix is stabilized by intrachain H bonds)
-hydrophobic interactions

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11
Q

Describe the first two laws of thermodynamics.

A

First Law of Thermodynamics- states that the total energy of a system and its surroundings is constant.
+requires that any energy released in the formation of chemical bonds must be used to break other bonds, released as heat or stored in some other form.

Second Law of Thermodynamics- states that the total entropy of a system plus that of its surroundings always increases.
-Gibbs free energy law

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12
Q

Understand free energy and chemical equilibrium.

A

Free energy (delta G) is the energy used to do work. A negative free energy change will occur if there is an increase in entropy. Reaction drivers include increase in concentration of the reactants over the product

ΔGo’=-RTlnKeq, Keq= products/reactants

G determines whether rxn will occur spontaneously and energy required determines the rate of reaction(thermodynamics). Take into account endergonic and exergonic.

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13
Q

Describe in general biological structures and metabolic processes.

A

-metabolism is composed of many coupled, interconnecting reactions
+mechanical work in muscle contraction and cellular movement
+active transport of molecules and ions
+synthesis of macromolecules and other biomolecules
-Metabolism is a linked series of chemical rxns that begin with a particular molecule and converts it into some other molecule(s) -> interdependent
-can divide into two classes: those that convert energy into useful forms (catabolism) and those that require energy to proceed (anabolism)
-must be thermodynamically favored and have a specific pathway
-the free energy for coupled rxns is equal to the sum of the individual steps, so a thermodynamically unfavored rxn can be driven by a couple favored rxn or have ATP drive it
-ATP-ADP cycle is the fundamental mode of energy exchange in biological systems
-ATP readily gives up its phosphate because the ADP has better resonance stabilization, has less electrostatic repulsion, and is stabilized due to hydration.
-molecules with higher phosphoryl-transfer potential than ATP:
+phosphoenolpyruvate(PEP)
+1,3-bisphophoglycerate (1,3-BPG)
+creatine phosphate
-these donate P to ADP

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14
Q

Understand principles associated with acids, bases, and buffers.

A

Transfer reactions
-oxidation-reduction reactions
-acid-base reactions: prominent way that biochemical processes occur
K= [H+][OH-]/[H2O]
Kw= 10^-14
-DNA helix begins dissociating at pH 9-10

The susceptibility of a proton to removal by reaction with base is described by pKa:
pKa= -log(Ka) -> Ka= [H+][OH-]/[H2O]
When pH=pKa, [A-]=[HA] and deprotonation process is halfway through
-buffers: resist pH changes
apply Hendersen-Hasselbach equation
pH=pKa + log([A-]/[HA])
-buffers function best closest to their pKa

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15
Q

Categorize the amino acids.

A

Essential:

  • histidine
  • isoleucine
  • leucine
  • lysine
  • methionine
  • phenylalanine
  • threonine
  • tryptophan
  • valine
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16
Q

Understand the special roles of nonstandard amino acids.

A

Nonstandard amino acids are naturally occurring, but don’t participate in peptide synthesis. They are constituents of peptides, but are modified after peptide synthesis. Typically occur as intermediates in metabolic pathways for standard amino acids.

Examples:

homocysteine
selenocysteine(found in genetic code)

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17
Q

Understand that, over time, a protein’s structure is more highly conserved than it’s sequence.

A

yay

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18
Q

What is the type of information that is available from bioinformatics databases and programs.

A

Bioinformatics is used to compare different proteins to determine the similarity between them. This is used to determine homology (ortholog v paralog).

  • this can occur via shuffling where the sequences are physically moved and the matched residues equal 10pts and gaps are -25pts. Add these values up to determine the percentage of identicality.
  • there can be conservative substitutions replacing one aa with one of similar size and chemical properties. To determine the probability that this occurred a substitution matrix (Blosum-62) is utilized. A positive score corresponds to a substitution that occurs frequently whereas a negative score happens less so. Detects homology between less obviously related sequences.
  • lack of statistically significant degree of sequence similarity does not rule out homology
  • BLAST sequences to detemine homology and figure out ancestors
  • tertiary structure is closer associated with function than structure therefore it is more conserved
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19
Q

Describe secondary and tertiary structure.

A

Secondary structure- the folding of the polypeptide chains into alpha helices, beta sheets, beta turns, or omega loop. These foldings are determined by the sequences that have characteristics that allow for hydrogen bonding. These sequences find each other and begin folding into one of the basic structures

Tertiary- the overall 3D shape of the protein. The bending and twisting of the protein to achieve lowest energy conformation (most stable). This is determined by hydrophobic regions, disulfide bridges, metal ions, and H bonds.

20
Q

What are the structural differences between fibrous and globular proteins.

A

Fibrous- consist of polypeptide chains arranged in roughly parallel fashion along a single linear axis, forming tough, usually water insoluble fibers or sheets. Ex. keratin, collagen, actin, myosin

  • alpha helices intertwine to form a super helix and the toughness depends upon the crosslinks
  • hydoxyl groups are added to collagen and in scurvy those are not added, so impossible to make collagen

Globular- many of the known enzymes, show a tightly folded structural geometry approximating the shape of an ellipsoid or sphere

21
Q

Describe heat induced protein denaturation.

A

Denaturation occurs when there are external conditions that will break the bonds holding the protein together such as temperature, pH, oxidizing/reducing agents, agitation (shaking), detergents, chaotrophic agents, organic solvents.
-chaotrophic agents dehydrate the protein and break the hydrogen bonds holding it together

22
Q

What are the features of quaternary structures.

A

Quaternary structures are multiple protein subunits that are held together with noncovalent bonds.

23
Q

Describe the GroEL/ES system.

A

These belong to the chaperonine family and is found in bacteria. Required for proper protein folding in bacteria. Requires 7 ATP to function. The unfolded protein enters the GroEL and ATP binds inducing a conformational change. The GroES caps the GroEL and more ATP is utilized to release the properly folded protein.

24
Q

Understand that molecular chaperones assist protein folding via ATP dependent bind and release mechanism.

A

ok

25
Q

Explain the Chou-Fasman method.

A

The Chou-Fasman method of pretending secondary structure of amino acids predicts the conformation based on the amino acids found of the chain and what affinity a majority of those amino acids have for which structure. The propensity/natural inclination can be calculated. See previous slide for calculations.

26
Q

Understand why nonpolar residues occur in the protein interior and polar residues on the exterior.

A

nonpolar residues are hydrophobic therefore they have a natural tendency to fold inward away from water and polar residues are hydrophilic and will prefer the aqueous environment. If these residues fold this way then the protein will have a low energy conformation and be more stable.

27
Q

Understand that a protein’s tertiary structure consists of secondary structural elements that combine to form motifs and domains.

A

yeppers

28
Q

Be able describe the protein misfolding that occurs in neurodegenerative diseases.

A

-termed prions
characteristics include:
-consists of aggregated forms of a specific protein, range of MW.
-resistant to treatment that degrade most proteins
-largely derived from a protein (PrP) that is normally present in the brain

  • normal PrP contains lots of alpha helix and little beta sheets, the infected form converts the alpha helix int beta strands. The beta strands of one protein link with another beta sheet joining the two proteins leading to the formation of aggregates.
  • fibrous aggregates are referred to as amyloid forms
  • Alzheimer disease have structures called amyloid plaques, prone to forming insoluble aggregates
  • theory states that large aggregates are not toxic, but smaller aggregates of the same protein damage the cell
  • can be transferred between species
29
Q

Understand the role of trace metals in protein folding.

A

-metal ions are needed by at least 1/4 of proteins
-metallochaperones insert the correct metal ions into the protein
+acquire metal ions directly from cellular pools
+can form more stable metal ion/protein complexes than others
-not clear what cellular mechanism enable a nascent protein to incorporate the correct metal ion
-heavy metals are inhibitors of protein folding (Cd, Hg, Pb)

30
Q

Be able to describe hemoglobin function.

A

Hemoglobin transports oxygen throughout the body by binding O2 with the four subunits that comprise the structure. Oxygen is bound by Fe+2 molculesO2 can be reversibly bound, controlled allosterically, and be cooperative.

31
Q

Understand the structural basis of hemoglobin.

A

-need iron, B12, B6, and folic acid for erythropoiesis
-RBCs live for 120 days
-composed of four identical subunits (tetramer) with globin folding
-96% composed of 2 alpha chains and 2 beta chains
-four heme groups that fix iron
-porphyrin ring and 4 Fe+2
-composed of 8 alpha helices that have two histidine residues
+proximal histidine (F8) is close to the iron, binding to it and creating a 5th ligand, causes conformational change which pulls Fe down to pyridine, causing a conformational shift in the tetramer. Helps promote the opening of the other binding sites for O2 (15degree rotation)
+distal histidine (E7) is utilized to outcompete CO affinity in heme by creating a channel for O2

32
Q

What is allosteric regulation in hemoglobin?

A
  • Allosteric regulation in hemoglobin includes 2,3-BPG where it is only present in the T state and is released in the R state bc the structure “collapses” the hole between subunits that 2,3-BPG occupies. There must be more oxygens bound to the heme group in order to induce T to R transition.
  • protons and Co2 also act as allosteric effectors
  • CO2 binds directly to the heme group switching it to the T state -> lower O2 affinity
  • protons lowers the pH of the blood (exercise) and decrease heme affinity for O2. Protonates histidine and amino termini.
  • Bohr effect: rapidly metabolizing tissues produce H+ and CO2, there will be more oxygen circulated to those tissues
33
Q

Understand hemoglobin cooperativity.

A

A tetrameric molecule that can exist in of two conformations, so the conformational change in one subunit induces a change in another subunit, switch and lock conformation

34
Q

What is meant by the term active site? What is its role in enzyme specificity?

A

The active site of the enzyme binds the substrate and undergoes the chemical reaction/alteration.

-the specificity of binding depends on the defined arrangement of atoms in the active site. The site typically only binds to one specific substrate explained via lock and key model or induced fit.

35
Q

What are the major coenzymes (including vitamins) and what type of reaction is associated with each?

A

-a cofactor is able to execute chemical reactions that cannot be performed by the standard set of 20aa.
apoenzyme + cofactor = holoenzyme
-coenzymes are small organic molecules that are either tightly (prosthetic group) or loosely bound to the enzyme

36
Q

Define what is meant by the term allosteric site and explain how an allosteric enzyme can be activated or inhibited.

A

-enzyme inhibitors that interfere with catalysis by slowing or halting enzymatic reactions
+competitive: inhibitor binds to active site blocking substrate (increases Km)
+uncompetitive: binds to opening of active site trapping substrate in active site (Km and Vmax altered)
+noncompetitive: binds to another site on the enzyme changing the conformation of the active site (affects Vmax)
-important to pharmaceuticals and helped map out the steps in a reaction

37
Q

What are the major enzyme groups and some characteristics of each?

A
  1. Oxidoreductases: oxidation-reduction (transfer electrons from one molecule to another)
    ex. alcohol dehydrogenase converts alcohols to aldehydes or ketones with reduction of NAD+ to NADH
  2. Transferases: catalyzes the transfer of a functional group from one molecule to another (donor molecules required)
    ex. kinase receptors phosphorylate proteins and themselves (serine, tyrosine, and threonine)
  3. Hydrolases: catalyzes the hydrolysis of covalent bonds (transfer functional groups to water)
    ex. dissacharidases- cleave double sugars into single sugars, defects in lactase (lactose intolerance)
  4. Lyases: catalyzes the addition or removal of groups to form double bonds
    ex. aldolase catalyzes second step of glycolysis
  5. Isomerases: catalyzes isomerization (intramolecular group transfer, product is an isomer of the substrate)
    ex. triose phosphate isomerase- intramolecular oxidation-reduction reaction
  6. Ligases: catalyzes the covalent linkage of two substrates (ATP hydrolysis)
    ex. DNA ligase forms two covalent phosphodiester bonds between nucleotides
38
Q

Know that enzymes are biocatalysts and speed up reaction rate without being consumed and produce the same amount of product.

A

good to know :)

39
Q

What is the Michaelis-Menton theory of enzyme kinetics? Be able to graphically determine Km and Vmax.

A
  • a model of enzyme kinetics describing the rate of enzymatic reactions by relating reaction rate to substrate concentration.
  • Vmax: maximum rate of enzymatic reaction
  • Km: substrate conc at half Vmax
  • sigmoid graph
  • Lineweaver-Burk is straight line ->
40
Q

Explain the effect of pH on enzymatic reactions.

A
  • enzymes have an optimal pH that helps maintain their 3D shape
  • changes in pH may denature enzymes by altering the enzyme’s charge (altering ionic bonds that contribute to functional shape
41
Q

Describe the kinetics of bisubstrate reactions.

A

-most biological reactions start with two substrates and end with two products
-transfer one functional group from one to another
-sequential reactions
+all substrates bind to the enzyme before any product is released
-double displacement reactions (ping-pong)
+one of the products is released before a second substrate binds

42
Q

Describe the catalytic process/mechanism employed by serine proteases in the hydrolysis of peptide bonds.

A

-use active site serine for peptide cleavage
-chymotrypsin active site
-preferred site-Phe- binds a hydrophobic pocket
+cleave backbone after Phe residue
Mechanism:
-nucleophilic attack of serine on peptide carbonyl group
-nucleophilic attack of water on acyl-enzyme intermediate
-release of carboxylic acid component

-activates zymogens by cleaving peptides (pepsinogen -> pepsin)

43
Q

Amyloid formation

A
  • cross beta structure with beta strands perpendicular to the backbone structure
  • polypeptide is derived from a cellular protein, amyloid precursor protein (APP
  • can be formed in vitro by many other peptides and protein
  • characterized by a log phase followed by a period of rapid growth (typical of nucleated processes such as crystallization)
44
Q

What are the characteristics of infectious proteins?

A
  1. transmissable agents are the aggregates of specific proteins
  2. protein aggregates are resistant to dissolving
  3. the protein is largely and completely derived from a cellular protein
    +PrP -> PrPsc
    +amyloid plaques -> b-amyloid
    +alpha synuclein -> plaques
    +SOD1 -> monomer SOD1 plaques
45
Q

What is the role of 2,3-biphosphoglycerate in red blood cells? Why would you have more at higher altitudes?

A

2,3-BPG lowers the hemoglobin’s affinity for oxygen as it carries it (loosely bound), so that it will be released into the tissues. Without 2,3-BPG only 8% of the oxygen carried by the blood would be released. You would have more at higher altitudes, so that more oxygen can be released into the tissues due to lower concentration of oxygen in the atm.

Biochemistry-Erin (2 decks)

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