Exam 2 Review Flashcards
Michaelis-Menten Equation / Plot
V_0 = Vmax[S] / K_m + [S]
Hyperbolic curve
- V0 = “initial rate” = product/time
- Vmax = maximum V0 at infinite [S]
- kcat = inherent turnover number of enzyme =
observed Vmax normalized to amount of
enzyme present = Vmax/[Et] - Km = [S] at 1/2 Vmax = same as Kd if k2
(chemical step) is slow relative to S binding - kcat/Km = catalytic efficiency of enzyme
Nucleotides
- Three components (base, phosphate, and ribose – a pentose)
- Two kinds of pentose: Ribose (2’ OH, RNA), deoxyribose (2’ H, DNA)
- Three general uses:
- Present in DNA and RNA
- Act as a source of energy for living systems
(ATP and GTP) - Cellular signaling
Nucleosides lack the phosphate group of
nucleotides
What is the role of cofactors in enzymatic reactions?
Cofactors participate in catalysis or stabilize protein structure.
Define coenzymes.
Organic or metalloorganic molecules that carry functional groups necessary for enzymatic activity.
Nucleotides: Use as coenzymes
* Many enzyme cofactors or coenzymes include adenosine
* Adenosine part does not directly participate in catalysis, but contributes binding energy
* Examples:
* CoA: transfers acyl groups
* NAD: carries electrons
* FAD: carries electrons
Polymerase Chain Reaction: PCR
The polymerase chain reaction (PCR) provides
a convenient and rapid method for amplifying
segments of DNA if the sequences of the ends of the targeted DNA segment are known.
What is the transition state in a chemical reaction?
The point at which the formation of substrate or product is equally likely to happen.
What does ΔG‡ represent?
The activation energy, the difference between ground state energy level and transition state energy level.
What does the ES complex represent?
The transient complex of the enzyme with the substrate.
Ribose conformations
- Conformation of pentose: four different “puckered” conformations (4 of 5 atoms in a single plane), C-2’ endo or exo, C-3’ endo or exo
- Different conformations found in different DNA/RNA structures
DNA sequencing: Sanger method
- Four reactions are run, containing:
- template DNA (to be sequenced)
- oligonucleotide (short DNA)
- DNA polymerase (enzyme) to make the DNA
- dNTPs (dATP, dGTP, dCTP, and dTTP)
- In each reaction, one of the four nucleotide pools includes some radioactive dideoxy NTP (ddNTP) chemically altered bases
- Lack of 3’OH in ddNTP halts DNA synthesis (replication)
- ssDNA strands can be separated with electrophoresis
- The DNA sequence can then be read off from the gel from bottom (smallest) to top (biggest)
- NB: this sequence is complementary to the original DNA strand
What does the Michaelis-Menten equation describe?
The relationship between reaction velocity and substrate concentration.
What does K_m indicate?
Substrate binding affinity; it is the same as the dissociation constant K_d.
What is k_cat?
The turnover number, the number of molecules of substrate converted to product per second at saturating substrate concentration.
How is catalytic efficiency represented?
As k_cat/K_m, indicating how effectively an enzyme converts substrate to product.
What happens to V_max and K_m during reversible competitive inhibition?
V_max remains the same
K_m increases
What type of enzyme is chymotrypsin?
A protease that catalyzes hydrolytic cleavage of polypeptide bonds.
- substrate specificity: peptide bond after aromatic aa (F, Y, W)
- nucleophilic attack of substrate by Ser195
- Ser195 reactivity tuned by adjacent residues
- acyl-enzyme intermediate
Chymotrypsin’s reaction?
- Substrate (polypeptide containing an aromatic residue) binds to chymotrypsin active site
- Aromatic side chain fits in hydrophobic pocket
- Same residue’s C=O fits in oxyanion hole
- His57 H-bonds with Ser195, making it a nucleophile
- Ser195 attacks the substrate C=O carbon, forming covalent tetrahedral intermediate
- Oxyanion hole, including Ser195 and Gly193 main-chain N—H, stabilizes negative charge on C—O-
- The unstable tetrahedral intermediate breaks down
- The part of the substrate polypeptide that was C-terminal to the aromatic residue leaves as product 1
- His57 protonates the amino leaving group
- A covalent acyl-enzyme intermediate is left over
- Now for the second half of the reaction…
- A water molecule (from the surrounding solvent) gets deprotonated by His57
- The resulting OH- attacks the substrate C=O carbon of the acyl-enzyme intermediate
- Forms a second covalent tetrahedral intermediate
- Oxyanion hole again stabilizes negative charge on C—O-
- Second tetrahedral intermediate also breaks down
- The part of the substrate polypeptide that was N-terminal to the aromatic residue, now with a new C-terminus, becomes product 2
- Product 2 dissociates
- The cycle is complete
- Chymotrypsin can catalyze another cycle of proteolysis
What stabilizes the negative charge on the tetrahedral intermediate in chymotrypsin’s reaction?
The oxyanion hole, which includes Ser195 and Gly193 main-chain N—H.
Irreversible enzyme inhibition
An irreversible inhibitor prevents this by
permanently modifying the enzyme
- Reaction of chymotrypsin with
diisopropylfluorophosphate (DIFP) leads
to a covalent modification of the
enzyme’s nucleophile (Ser195) to
irreversibly inhibit the enzyme - Ser195 can’t carry out its function, thus
the enzyme is no longer active
What does the Lineweaver-Burk plot help to calculate?
V_max and K_m from the x- and y-intercepts.
What type of intermediate is formed when His57 protonates the amino leaving group?
Covalent acyl-enzyme intermediate
This intermediate is crucial for the reaction mechanism involving His57.
What role does water play in the second half of the reaction? Chymotrypsin
Deprotonated by His57 and attacks the substrate C=O carbon
This step leads to the formation of a second covalent tetrahedral intermediate.
How does pH affect enzyme activity?
-
pH alters ionization of R groups, coenzymes,
and cofactors which in turn may alter the 3D
structure of enzyme or chemistry of active site - often pH optimum is related to biological
setting: -
pepsin is a digestive enzyme in stomach
(pH optimum ~ 1.6); pH in gastric juice is ~
1.5 after eating a meal -
glucose 6-phosphatase acts in liver cells
(pH optimum ~ 7.8); pH in cell ~ 7.2
This can change the 3D structure of the enzyme or the chemistry of the active site.
Define allosteric enzymes.
- Allosteric enzymes have separate binding
sites for molecules that can affect catalysis at
the active site - Regulation can be positive or negative
- Analogy to H+, CO2, and BPG for hemoglobin
(though Hb is not an enzyme) - Allosteric modulator can be same as substrate (homotropic) or different from substrate (heterotropic)
- Many allosteric enzymes have multiple subunits (e.g. quaternary structure)
- Each subunit may bind substrate and/or positive and/or negative allosteric modulators
They can exhibit positive or negative regulation.
Aspartate transcarbamoylase (ATCase)
Allosteric enzyme
- 12mer with many binding sites
- Catalyzes early step in production of pyrimidines (T and C in DNA)
- CTP binds to an allosteric site as a negative allosteric regulator
- “feedback inhibition”
- when plenty of pyrimidines available
- ATP binds to another allosteric site as a positive allosteric regulator
- when the cell is happily growing, has plenty of energy, and may need more pyrimidines
These modulators influence enzyme activity in different ways.
What is the characteristic curve of allosteric enzymes?
Sigmoid instead of hyperbolic
This indicates a cooperative binding effect.
Allosteric enzyme: control
- “+” = positive allosteric modulator; lowers
K0.5 - “-” = negative allosteric modulator;
increases K0.5 - Vmax doesn’t change here, but can in
other cases
Ribose is a pentose sugar that can be either ribose or deoxyribose.
What is the difference between ribose and deoxyribose?
Ribose has a 2’ OH, deoxyribose has a 2’ H
This difference is crucial for the structure of RNA and DNA.
What are the two parent compounds of nucleobases?
- Pyrimidine (1 ring CTU)
- Purine (2 rings AG)
- Weak bases
- Aromatic molecules
- Most of the bonds have double bond character
- Adenine (A), Guanine (G), and Cytosine (C) are present in DNA and RNA
- Thymine (T) is only present in DNA; Uracil (U) is a demethylated form of thymine and takes its place in RNA
- Bases (Pu/Py) have highly conjugated structures that absorb in UV ~ 260 nm
- Free bases are poorly water soluble at neutral pH, more soluble in acid/base (more charged)
These bases are essential components of nucleotides.
What type of bond is formed by the removal of water in nucleobases?
N-β-glycosyl bond
This bond connects the base to the sugar.
DNA and RNA
- Nucleic acids are polymers of nucleotides
-
Phosphodiester bonds link successive nucleotides in
nucleic acids: the phosphate group “bridges” the 3’-OH
of one nucleotide to the 5’-OH of the next - DNA (deoxyribonucleic acid) lacks an OH group at C2’
- RNA (ribonucleic acid) possesses an OH group at C2’
- The phosphates have a pKa ~ 0! They always have a negative charge and form complexes with proteins and divalent cations (e.g. Mg2+)
- The ends of nucleic acids are referred to as 5’ and 3’. This gives them directionality
- DNA/RNA sequence convention is 5’ to 3’:
- 5’-GAATTC-3’ (phosphate at 5’, hydroxyl at 3’
- Both DNA and RNA undergo slow nonenzymatic hydrolysis of the phosphodiester bond in vivo under alkaline condition
- RNA is less stable than DNA because of the ribose 2’ OH group
DNA structure: The double helix
- Primary structure = nucleotide sequence
- Secondary structure = right-handed helix
- Tertiary structure = folding in chromosomes (structure becoming understood).
- Hydrogen bonding between specific bases is preserved (not so with RNA)
- Hydrogen bonding occurs between carbonyls and protonated nitrogens
- Note: A-T has 2 H-bonds, but G-C has 3 H-bonds = stronger
These bonds connect the phosphate group of one nucleotide to the 3’-OH of another.
DNA Structure
- 36 Å per repeat of complete turn, 3.4 Å distance between stacked bases per bp rise, 10.5 bp (base pair) per turn
- Structural stability due to:
- Hydrophobic interactions among stacked bases pi-bonding overlap = most important
- H-bonds between complementary bp = less important for stability
DNA structure: Torsion angles
- Rotation about seven different bonds (w/ limited rotation about bond 4) connecting sugar (ribose) and phosphate
- Limited rotation about bond 4 gives rise to ring pucker (endo: cleaves internally vs. exo: cleaves externally)
- DNA sequence can affect these angles. This gives the DNA a particular structure that may be important for protein binding; thus gene regulation
What are the three structures of DNA discovered to date?
- A-form: believed to be a chemical artifact as a
result of dehydration of B form - B-form: most DNA in vivo
- Z-form: Found in crystals near promoters (possibly responsible for gene regulation or genetic recombination?)
- Z-DNA favored by alternating purines-pyrimidines (Pu-Py) steps
- Pyrimidines remain anti, but purines “flip” from anti to syn, so sugar-P backbone “zig-zags”
(hence “Z-DNA”)
A-form: believed to be a chemical artifact as a result of dehydration of B
form
B-form: most DNA in vivo
Z-form: Found in crystals near promoters (possibly responsible for gene regulation orgenetic recombination?)
DNA origami and nanostructure
Palindromes read same in forward and reverse
* In DNA, a palindrome is split across complementary strands
* Can lead to the formation of hairpins in ssDNA or RNA.
* Sites recognized by many DNA binding proteins (i.e. restriction enzymes)
* Hairpins and cruciforms are self-complementary and have been seen in vitro, yet rarely in vivo
Hoogsteen base pairing & alternative structures
- Hoogsteen base pairs (red, right) allows for the formation of triple-helical DNA with two pyrimidine strands and one purine strand and parallel or G tetraplex strands.
- G tetraplex (four strands DNA) occurs only when many guanosine bases present, including telomeres at the end of chromosomes
- Can be parallel or anti-parallel
What is the function of mRNA?
Codes for polypeptide chains
Formed on a DNA template by the process of transcription
• mRNA (messenger RNA) codes for polypeptide chains: monocistronic (one gene; prokaryotes and eukaryotes) or polycistronic (multiple genes; prokaryotes). Cistron is the genetics definition of a gene. Eukaryotic genes include mix of protein-coding segments (exons) and non-coding segments (introns).
• Untranslated regions (UTRs) serve for protein binding sites, important for regulation of translation, mRNA stabilization
It serves as a template for protein synthesis.
What is the role of tRNA in protein synthesis?
Essential for mRNA translation
• Nearly all tRNA are L-shaped to fit into the ribosome (three-leafed clover)
• AA gets pre-loaded into acceptor loop
• Anticodon loop must base-pair with mRNA codon
• A codon is a DNA or RNA sequence of three nucleotides that forms a unit of genomic information encoding a particular amino acid.
• Each codon represents a particular amino acid, and each codon (3bp) is recognized by a specific tRNA
It carries amino acids to the ribosome based on codon recognition.
What is a codon?
A sequence of three nucleotides encoding a specific amino acid
Each codon corresponds to a specific tRNA.
What are the primary structures of polysaccharides?
- Polysaccharides may be composed of one, two, or several different sugars in straight or
branched form - 19 common monosaccharides act as building
blocks of complex polysaccharides - Monosaccharides are attached to one another by a series of enzymes
- Unlike protein, no template or defined MW. Synthesis program is intrinsic to the Enzyme
- Also unlike protein & nucleic acids, polysaccharides are NOT identical – they are quite heterogeneous
This forms the backbone of polysaccharides.
Secondary structures of polysaccharides
- van der Waals interactions, H-bonding, and ionic forces influence the polysaccharide fold (similar to proteins)
- Dihedral angles φ and ψ can be used to describe the 3D relationship between two adjacent saccharides (similar to the Ramachandran plot). Free rotation C-O bonds linking the residues
- Lower energy conformations are favored (similar to proteins)
- Dihedral angles can be used to describe the “fold” of a polysaccharide (similar to proteins)
ie: starch (amylose): polymer of many D-
glucose units in α1→4 linkage
* its chair conformation leads to a curved
(180°) and tightly coiled helical structure (most stable)
* this structure excludes water and is responsible for dense granules present in cells
* Amylose components interact heterogeneously with one another, causing the formation of a coiled helical structure
This formula represents the basic composition of carbohydrates.
Starch vs Cellulose
Starch (storage of energy) Glucose alpha-linked polymer
* contains two types of D-glucose polymers (in chloroplasts: ~0.4 M)
* Amylose (MW: ~103 to 106 Da; linear α1→4) and amylopectin (MW: few million Da; linear
α1→4, branched at α1→6) — each with one reducing end
* Amylose and amylopectin form starch granules in cells
* Degradative enzymes act at nonreducing ends, thus branches allow more rapid degradation
* Storage of glucose as polymers decreases osmolarity. [Glucose] ~ 0.4 M would cause water to enter cell, cell would swell and lyse
Cellulose: Glucose beta-linked polymer
* linear polymer of D-glucose
units with β1→4 linkages (—>)
* Glucose has β configuration
* Polymers adopt extended structures that pack against one another, similar to strands in β-sheet
* Cellulase: enzymes secreted by bacteria in the stomach of termites
* Chitin: differs from cellulose by replacement of OH at C-2 with an acetylated amino group (N-acetyl glucosamine units)
* Chitin is indigestible for vertebrates (lack chitinase enzymes)
* Forms exoskeleton of insects, lobsters, etc.
* Second most abundant polysaccharide, after cellulose
What are the two most common monosaccharides?
- Glucose
- Fructose
- monosaccharides except dihydroxyacetone have asymmetric (chiral) carbon atoms
- stereoisomers are non-superimposable
- there are 2N stereoisomers for N chiral centers
- chiral assignment: Chiral center most distant from most oxidized carbon is compared to D/L
glyceraldehyde. D- when hydroxyl is on right,
and L- when hydroxyl is on left
These sugars are fundamental to biological processes.
Large RNPs: RiboNuclear Protein machines (Ribosomes)
A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the mRNA sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.
Hybridization: How DNA/RNA strands find each other
- Hybridized DNA and RNA can be denatured and renatured. The melting temperature (Tm) depends upon the GC composition and degree of complementarity between strands. Disruption of the H-bonds is reversible.
- ssDNA to dsDNA (annealing) begins slowly by random collisions, then when in register, it “zips” up
- Central process to nucleic acid biochemistry and analytical processes
Mutagens & mutagenesis
- Some well-characterized nonenzymatic reactions of nucleotides occur very slowly and lead to mutations; perhaps a link to aging and carcinogenesis.
- Many corrected by DNA repair system, but not 100% fidelity!
Nucleotides: Use for energy currency and signaling
- Nucleoside Triphosphates (NTPs) are precursors of DNA/RNA
- NTPs also store energy (ATP/GTP)
- Hydrolysis of an anhydride bond yields more energy than hydrolysis of the ester
- ATP hydrolysis provides energy
for biosynthesis - ATP = “energy currency”
Each codon corresponds to a specific tRNA.
What is the significance of carbohydrates in photosynthesis?
Convert CO₂ and H₂O to cellulose and other carbohydrates
This process is critical for energy production in plants.
What defines a chiral center in monosaccharides?
Asymmetric carbon atoms
Chiral centers lead to stereoisomers.
How is chiral assignment determined?
By comparing the chiral center to D/L glyceraldehyde
D is when the hydroxyl is on the right, L is when it is on the left.
What is a chiral center?
A carbon atom that is attached to four different groups, creating non-superimposable mirror images.
How is the D/L assignment determined for sugars?
D- when the hydroxyl is on the right and L- when the hydroxyl is on the left compared to D/L glyceraldehyde.
D-aldoses and D-ketoses
- In all these D-isomers the chiral carbon most distant from the carbonyl carbon has the same
configuration as the chiral carbon in D-glyceraldehyde (OH on the right hand side.)
Cyclic forms of monosaccharides
- the hemiacetal or carbonyl carbon atom is called the anomeric carbon*
- An anomeric carbon is a carbon atom in a sugar that becomes a stereocenter when the sugar forms a ring
- isomeric forms of monosaccharides that differ only in their configuration at the hemiacetal or hemiketal carbons are
“anomers” - α if substituents of anomeric carbon (OH) and last stereocenter carbon (C5) are on opposite sides of ring
- β if they are on same side of ring
- mutarotation - interconversion of α and β anomers in solution (equilibrium mixture with 1/3 α, 2/3 β and small amount
liner )
What are epimers?
Two sugars that differ in configuration at only one carbon.
Which stereoisomers are more common, D- or L-?
D- stereoisomers are more common than L- stereoisomers.
What reaction occurs between the aldehyde group at C-1 and the hydroxyl group at C-5 in carbohydrates?
It forms a hemiacetal linkage producing either α or β anomers.
What is the dominant form of monosaccharides in aqueous solution?
The cyclic form.
What is the anomeric carbon?
The carbon atom in a sugar that becomes a stereocenter when the sugar forms a ring.
What defines an α anomer?
The substituents of the anomeric carbon (OH) and last stereocenter carbon (C5) are on opposite sides of the ring.
What defines a β anomer?
The substituents of the anomeric carbon (OH) and last stereocenter carbon (C5) are on the same side of the ring.
What is mutarotation?
The interconversion of α and β anomers in solution.
-OH swaps position
What is the difference between pyranose and furanose forms of sugars?
Pyranose (6C-ring) and Furanose (5C-ring) forms of D-glucose and D-fructose
- Pyranoses (6-C ring) interconvert between two possible chair conformations at room
temperature, with no bonds breaking or forming, making this a conformational change (instead of configurational). - Bonds to the substituents and hydrogen atoms on the ring carbons are axial (ax) or equatorial (eq).
- Also in “boat” conformation
This P looks like 6
F in furanose stands for 5
What is maltose?
A disaccharide formed when C-1 (anomeric) –OH of one glucose condenses with C-4 –OH of another glucose. Hydrolysis
- Result: elimination of H2O, formation of O-glycosidic bond, conversion of hemiacetal to acetal (add another alcohol)
- Resulting disaccharide has a “reducing end (3’end with -OH group)” at the remaining anomeric carbon which can be readily oxidized (in the linear form)
- Glycosidic bonds are hydrolyzed by acid (but resistant to base), so boiling in dilute acid generates free monosaccharides
What is a glycosidic bond?
A bond formed between two monosaccharides during the condensation reaction.
What are the primary structures of polysaccharides?
Polysaccharides may be composed of one, two, or several different sugars in straight or branched form.
What distinguishes polysaccharides from proteins and nucleic acids?
Polysaccharides are not identical and are quite heterogeneous.
How do van der Waals interactions and H-bonding influence polysaccharides?
They influence the polysaccharide fold, similar to proteins.
What are the two types of D-glucose polymers in starch?
- Amylose (linear α1→4)
- Amylopectin (branched α1→4 and α1→6)
Sugar modifications
- Amino sugars: An NH2 group replaces
one of the OH groups in the parent hexose. - Deoxy sugar: H replaces an OH.
- Acidic sugar: COO- replaces a C—OH
What are glycosaminoglycans (GAGs)?
Long, unbranched polysaccharide chains composed of repeating disaccharide units.
• Glycosaminoglycans (GAGs): present in extracellular matrix (material/glue between cells) no in plants.
• GAGs are long, unbranched polysaccharide chains composed of repeating disaccharide units
• copolymers of disaccharides containing amino sugars and often modified with sulfates, amines, acetyls, …
• proteins (collagen, elastin. fibronectin, laminin) are interlocked by GAGs
• GAGs serve as receptors for a variety of molecules, proteins, pathogens…(electrostatic binding)
What is hyaluronate?
A viscous polymer present in the eye, cartilage, and tendons.
What are proteoglycans?
Proteins with GAGs attached, playing roles in the extracellular matrix.
• Proteoglycans consist of a core protein attached to one or more GAGs
• Many types of proteoglycans within an organism
• The glycans possess unique structures that correspond to unique chemical information
• Identical copies of a polypeptide chain can have different GAG compositions in different cell type
• Cells take advantage of this and make themselves unique to what molecules they interact with
• Cancerous cells have dysregulated GAGs on their proteoglycans
What distinguishes glycoproteins from proteoglycans?
Glycoproteins have smaller, branched glycans and do not have the tetrasaccharide linker.
What are the two types of glycosidic linkages in glycoproteins?
• O-linked: glycosidic link from anomeric carbon to -OH of amino acid side chains of Ser/Thr
• no specific sequence preference
• N-linked: N-glycosyl link from anomeric carbon to the amide side chain of Asparagine
• preference for Asn—nonPro-Ser/Thr
• Enzymes modify proteins with sugars
• Not all sequences are modified
• Aid with protein folding and stability
• Tissue specific glycoforms
What is the dominant surface feature of the outer membrane of gram-negative bacteria?
Lipopolysaccharides.
What is the fluid mosaic model?
A model describing the dynamic nature of membrane composition and architecture.
What is the difference between saturated and unsaturated fatty acids?
Saturated: higher melting point, solid at room temperature (esp. cis unsaturated) due to better Van der Waals interactions
* Great flexibility around each carbon, most favorable fully extended form.
Unsaturated: lower melting point, liquid at room temperature
* double bonds commonly between C9, C10 (∆9); others often ∆12 and ∆15
* Kink in hydrocarbon chain= they can not pack together as saturated fatty acid (d)
* cis fatty acids are far more common than trans fatty acids – trans found in bacterial fermentations and some dairy products; human lipases appear to be unable to metabolize
butter: 66% saturated : 33% unsaturated — solid
canola oil: 7% saturated : 93% unsaturated — liquid
What is the significance of the kink in unsaturated fatty acids?
It prevents them from packing tightly together, affecting their state at room temperature.
What are glycerophospholipids also known as?
Phosphoglycerides
What components make up glycerophospholipids?
Occur largely in the outer part of PM
- Tails: acyl chains (from fatty acids)
- Backbones: glycerol (C3) carbohydrate, with ester linkages to two acyl chains + phosphoester linkage to head group
- Head group: phosphate + groups at left
What character does sterols have?
Mixed polar (head group) / hydrophobic character
Cholesterol is important as a structural component and precursor of a variety of steroids
What is the lipid bilayer thickness?
- Lipid bilayer is ~3-4 nm (~30-40 Å) thick
The”bilayer thickness” is an average thickness (that reflects the local variation) that is different when proteins are embedded in the bilayer (60-100 Å). - cell walls are thicker than that because of the embedded transmembrane proteins, multiple types of lipids, and sterol
Differentiate micelles, bilayers and liposomes (vesicles)
- Micelles possess lipids with heads
having larger cross section than
the side chains - Bilayers possess lipids with heads
having same cross section as the
side chains - Liposomes (vesicles) = spherical
bilayers that have curved to close
their open ends
What are the three types of membrane proteins?
- Peripheral: proteins associate with membrane via electrostatic interactions, etc
- Integral: proteins are embedded into the membrane, or covalently modified via attachment of a lipid; can be removed by detergents
- Amphitropic: proteins reversibly associated with the membrane; regulated by covalently attached lipid anchors or ligands that induce a conformational change to expose a hydrophobic patch
Integral membrane proteins
6 types that use helices to span membrane
(H-bonds in helix made strong by acyls):
I. spans the membrane once, and its N-terminus is outside of the cell
II. spans the membrane once, and its C-terminus is outside of the cell
III. spans the membrane multiple times with termini at either end
IV. multiple polypeptide chains assemble to span the membrane multiple times
V. modified by GPI anchor, located inside the cell, and does not span membrane
VI. spans the membrane and possesses GPI anchors
Fluid mosaic model of membranes
- fluid: noncovalent interaction allows lipids and proteins to freely diffuse bilaterally
- mosaic: patchy surface (lipids and proteins
- proteins held in membrane by hydrophobic interactions with acyl portions of lipid
- proteins can diffuse across membrane -some proteins need to oligomerize for function
- membranes are impermeable to most charged and polar solutes
- functional asymmetry: extracellular face is decorated differently than cytoplasmic face — even the lipids themselves
Composition of membranes
- membranes are composed of many different types of lipids
- these lipids are spread unevenly throughout the membrane (intra- and inter-leaflet)
- the lipid composition is unique to organelle, cell, and tissue types
- lipid composition regulates the bioenergetics of the organelle, cell, and tissue type
Architecture of membranes
Organelles:
* mitochondria do ATP synthesis
* lysosome is the recycling center of the cell
* nucleus contains the genetic information
* rough and smooth ER synthesize protein and lipid, respectively
* Golgi processes proteins for secretion
What does a hydropathy plot display?
Hydrophobic and hydrophilic tendencies of an amino acid sequence
- the “smoothed” (window-averaged) hydropathy values are plotted as a function of residue
- extended regions that have positive hydropathy values (hydrophobic) are predicted to span the membrane
- only applicable to helices, not sheet
Helical membrane protein structure
- Aquaporin: transports water across membranes
- Protein structure is enveloped by phospholipids
- Hydrophobic interactions keep the nonpolar amino acid residues firmly anchored among the fatty acyl groups of the membrane lipids
What can happen to the physical state of a membrane?
- Membrane plasticity: change shape without losing their integrity
- Noncovalent interactions among the lipids allow their freedom of movement
- the physical state of a membrane can be altered by temperature or by its lipid composition
- liquid-ordered (L_0 at physiological T)⟷ liquid-disordered ( L_d above physiological T
Transbilayer
- lipids slooowly diffuse between monolayers (transbilayer)
- lipids quickly diffuse within a monolayer (lateral diffusion)
- enzymes can expedite transbilayer
translocation (flippase/floppase/scramblase) - flippases keep phosphatidylserine (PS) away from outer monolayer, where they would induce apoptosis!
What are lipid rafts?
- the surface of biological membranes are believed to be patchy
- these patches are called lipid rafts as they resemble rafts floating across an ocean
- lipid rafts have different components than their surroundings:
- enriched in sphingolipids and cholesterol
- are thicker (longer Fatty Acid chains of sphingolipid) and less fluid (result of cholesterol)
- enriched in particular proteins
Membrane fusion
- making one bilayer out of two bilayers
- the fusion of two membranes is central to a variety of cellular processes:
1. Golgi organelle: packages proteins to be embedded into peripheral membrane
2. exocytosis: release of cellular material into environment
3. endocytosis: uptake of extracellular material from environment
4. viral infection
5. fertilization
6. cellular division
What is GPI in the context of membrane proteins?
Glycosylphosphatidylinositol: a lipid + carbohydrate anchor covalently attached to some proteins
What types of transport are seen across membranes?
- Simple: ie O2 and CO2 for breathing
- Facilitated: ie glucose
- Primary active: ie Na+
What drives primary active transport?
Membrane proteins
What is the electrochemical gradient?
- the system comes to equilibrium by solutes moving down a concentration and electric gradient
- the combination of both is called electrochemical gradient or electrochemical potential (e.g. H+ moving into or out of the cell)
What’s functions of the electrochemical gradient?
- responsible for creating action potential in neurons
- allows selective diffusion of K+ (bigger radius: 1.33Å)
to pass 1x104 faster than Na+ (smaller radius: 0.95Å) down electrochemical gradient - negatively charged residues at entrance and exit increase the Na+ and K+ concentration
- negative dipole moment of helix also stabilizes cation
- hydration layer stripped upon entering selectivity filter
- the carbonyl backbone can stabilize K+, but too far apart to stabilize Na+ = basis for selectivity
- four binding sites along channel axis
- electrostatic repulsion between K+ places them in alternating binding sites
- the electrostatic repulsion from the incoming K+ drives them out of the cell by hopping one binding site at a time
What differentiates a transporter from an ion channel?
Transporter
* Two gates (open one at a time)
* Can move against the concentration gradient
Ion Channel
* The gate is either open or close
* Movement of ions limited by rate diffusion
* Flow down to the electrochemical gradient
* Biological signal can regulate the gate
A transporter protein stabilizes a transition state, speeding movement through a membrane
What are the types of transporters?
- Uniport: one molecule transporter
- Symport: two types of molecules in the same direction
- Antiport: two types of molecules in opposite directions
Can be active or passive
A co-transport system
- a chloride-bicarbonate exchanger
- present in erythrocyte membrane, responsible for solubilizing CO2 generated by respiring cells
- CO2 enters cell via simple diffusion across membrane
- carbonic anhydrase catalyzes formation of bicarbonate
- antiporter allows for diffusion of HCO3- out of cell only if Cl- can come into cell. Cl- is needed to keep the net charge inside the cell to 0 (H+) Electroneutral
- HCO3- can then diffuse thru the blood
Transporter: Ex. of facilitated passive diffusion
- cells need glucose for energy
- blood carries ~5 mM of glucose
- rate of glucose transport through transporter is 50,000x faster than through membrane
- GLUT1 (12 types, a uniport), 45 kDa, with 12 helical
- molecular mechanism of transport is modeled after kinetic studies:
- carrier can be saturated
- the reaction follows Michaelis-Menten kinetics behavior
- glucose in cell is quickly used
- no accumulation of glucose in cell
What happens to V_max and K_m during reversible uncompetitive inhibition?
V_max and K_m decrease
Permeability of Common Species
Low: Na+, K+, Cl-
High: Glucose, Thryptophan, Glycerol
What happens to V_max and K_m during reversible mixed inhibition?
V_max decreases and K_m goes either up or down
Allosteric enzymes
Allosteric enzyme - curve and K0.5
Which of the following aspects of a biochemical reaction is changed by an enzyme?
a. Free energy (G) of the substrate
b. Free energy (G) of the product
c. ΔG’° for the conversion of S -> P
d. ΔG‡ for the conversion of S -> P
e. K’eq for the conversion of S -> P
ΔG‡ for the conversion of S -> P
enzymes speed up reactions by lowering the activation energy (ΔG‡), making it easier for reactants to reach the transition state.
Which of the following is NOT an assumption of the Michaelis-Menten model of enzyme kinetics?
a. Reversion of product back to substrate is negligible
b. The accumulation of product is negligible
c. Substrate concentration is higher than enzyme concentration
d. The enzyme:substrate complex never dissociates
e. The enzyme:substrate complex is in a “steady state”
The enzyme:substrate complex never dissociate
Which type of enzyme inhibition can be overcome by increasing the substrate concentration?
Competitive inhibition
Uncompetitive inhibition
Mixed inhibition
Irreversible inhibition
All of them
Competitive inhibition
The Michaelis constant Km is equal to the dissociation constant Kd under what circumstances?
A) kcat is greater than Km
B) The rate constant for the chemical step of enzyme catalysis is very slow relative to association and dissociation of the enzyme:substrate complex
C) The concentration of substrate is much greater than Km
D) The concentration of substrate is equal to the concentration of enzyme
E) None of these answers is correct
The rate constant for the chemical step of enzyme catalysis is very slow
relative to association and dissociation of the enzyme:substrate complex
You are studying an enzyme of interest. You perform an enzyme activity assay at a variety of pH conditions, and observe that its maximum kcat and minimum Km are at pH 7. Which of the following is true?
A. It functions efficiently at physiological pH
B. It may have a His in the active site
C. It may have a non-His amino acid in the active site whose pKa is shifted
D. A decrease in pH due to cellular metabolism would decrease, not increase, enzyme activity
E. All of the above
E. All of the above
A. It functions efficiently at physiological pH
B. Since the enzyme’s maximum kcat and minimum Km occur at pH 7, this suggests that it
is optimized for function at or near physiological pH
C. It may have a His in the active site
Histidine has a pKa around 6.0, meaning it can be protonated or deprotonated around
neutral pH, making it a common residue involved in catalysis in enzymes that function near
pH 7.
D. It may have a non-His amino acid in the active site whose pKa is shifted
There are other residues that can be involved ( ex D, E…)
A. A decrease in pH due to cellular metabolism would decrease, not increase, enzyme activity.
the enzyme is most active at pH 7, a decrease in would likely lead to a reduction in
enzyme activity.
E. All of the above
Which of the following is FALSE about nucleic acids?
A. Polynucleotides are described in the 5’ to 3’ direction
B. RNA is more stable than DNA because it has a 2’ OH
C. Nucleic acids are made up of 3 main parts: nucleobase, sugar, phosphate
D. Divalent cations complement the negative charge of the phosphate backbone
E. All of these are true
RNA is more stable than DNA because it has a 2’ OH
Which of the following is true about the structure of DNA?
A. It predominantly adopts the A-form
B. The double helix is left-handed
C. Purines pair with purines and pyrimidines pair with pyrimidines
D. The structure is sequence-dependent
E. Base pairing is more important for stability than is base stacking
The structure is sequence-dependen
Which of the following is FALSE?
A. Monosaccharides are linked by O-glycosidic bonds to create
polysaccharides
B. Atom numbering starts at the anomeric carbon in aldoses
C. Alpha and beta anomers cannot interconvert
D. Phi and psi dihedral angles define polysaccharide secondary structure
E. Formation of a glycosidic bond is a condensation reaction
Alpha and beta anomers cannot interconver
How are proteoglycans and glycoproteins different?
A. Glycoproteins have a tetrasaccharide linker
B. Glycoproteins have longer, more negatively charged glycans
C. Glycoproteins have glycans attached at Ser-Gly-X-Gly motifs
D. Proteoglycans have glycosaminoglycans (GAGs) attached
E. None of the other answers are true
Proteoglycans have glycosaminoglycans (GAGs) attached
Which of the following is true?
A. Saturated fatty acids have cis rather than trans double bonds
B. Membranes form primarily because of enthalpic effects
C. The proportions of different lipid types in membranes are conserved across species
D. The center of bilayers are hydrophobic and the outsides are hydrophilic
E. Glycerophospholipids have several rings in their structure
The center of bilayers are hydrophobic and the outsides are hydrophilic
You are interested in studying membrane dynamics experimentally. To do so, you expose E. coli cells to a membrane-impermeable fluorescent probe that binds to lipid head groups. You then expose a specific region of the membrane to a laser pulse that deactivates the fluorescent probe. What result would you expect
to see?
A. Fluorescence disappears in the targeted region, then slowly reappears.
B. Fluorescence disappears in the targeted region, and does not reappear.
C. Fluorescence disappears throughout the entire membrane.
D. Fluorescence disappears in the targeted region, then slowly reappears — but
only in lipid rafts.
E. None of the above
Fluorescence disappears in the targeted region, then slowly reappears
What substrate characteristics are best for rapid transport across a membrane?
A. small, charged
B. small, uncharged
C. large, charged
D. large, uncharged
E. None of the above
small, uncharged
FRAP
- Fluorescence Recovery After Photobleaching
- Fluorescence disappears initially because of laser
pulse - Fluorescence returns after laser pulse because
lipids in membranes are dynamic
