CELL BIO: PROTEIN FUNCTION

What is a ligand-binding site?

A region of a protein that accepts and interacts with another molecule through multiple noncovalent interactions.

How does folding create a functional binding site?

It brings amino acids far apart in sequence into the correct three-dimensional positions; determines protein function

What three types of functional surfaces can folding create?

Enzyme active sites, receptor-binding sites, and protein–protein interaction surfaces.

What is an active protein conformation?

A folded shape in which the protein can carry out its function.

Which residues are listed as able to participate in ligand binding or catalysis?

Asparttic Acid D, glutamate E, arginine R, lysine K, histidine H, asparagine N, glutamine Q, serine S, threonine T, tyrosine Y, and cysteine C.

What ligand is shown in the binding-site example?

Cyclic AMP (cAMP).

Which residues are in the cAMP-binding-site example?

Serine, glutamate, arginine, and threonine.

What interactions stabilize cAMP binding in the example?

Hydrogen bonds and electrostatic attractions from appropriately positioned side chains.

Why can many weak interactions produce strong binding?

Their combined favorable binding energy gives the protein high affinity for the ligand.

How do different side chains contribute to ligand binding?

Their charges and other chemical properties support different noncovalent interactions.

What is induced fit?

Ligand binding causes a protein to change shape slightly, improving the fit and interactions.

What three residues make up a serine protease catalytic triad?

Aspartate, histidine, and serine (Asp–His–Ser).

How does the catalytic triad activate serine?

Hydrogen-bond rearrangements allow histidine to remove a proton from serine, making serine more reactive.

How can the Serirne protease catalytic triad residues cooperate if they are separated in sequence?

Protein folding brings them together in the active site.

What does high-affinity binding need to resist?

Thermal agitation, which can disrupt individually weak contacts.

What is a binding equilibrium constant measuring?

(Binding strength) The balance of bound and unbound states; for an association constant Kₐ, a higher value means stronger binding.

How does Kₐ relate to binding affinity?

Higher association constant Kₐ means higher affinity; the dissociation constant Kd has the opposite relationship.

How does more favorable binding energy affect Kₐ?

A modest favorable energy change can raise Kₐ substantially because the relationship is exponential.

What effect can adding or removing one or two hydrogen bonds have?

It can change a binding equilibrium constant by roughly tenfold or more, depending on the context.

What do enzymes change about a reaction?

They increase the reaction rate by lowering its activation-energy barrier.

Do enzymes change the reaction's equilibrium constant or overall ΔG?

No; they accelerate approach to equilibrium without changing its position or overall ΔG.

What is a hydrolase?

An enzyme that cleaves a bond by hydrolysis, using water.

What is a nuclease?

An enzyme that cleaves nucleic acids such as DNA or RNA.

What is a protease?

An enzyme that cleaves peptide bonds in proteins.

What is a synthase?

An enzyme that catalyzes synthesis or formation of a molecule. (anabolic reaction)

What is an isomerase?

An enzyme that rearranges atoms or bonds within a molecule to form an isomer.

What is a polymerase?

An enzyme that builds a polymer; DNA and RNA polymerases synthesize nucleic acids.

What is a kinase?

An enzyme that adds a phosphate group, often from ATP, to a substrate.

What does a protein kinase do?

Phosphorylates a protein.

What does a phosphatase do?

Removes a phosphate group by hydrolysis.

What is an oxidoreductase?

An enzyme that catalyzes an oxidation–reduction reaction in which electrons are transferred.

Name types of oxidoreductases.

Oxidases, reductases, and dehydrogenases.

What does an ATPase do?

Hydrolyzes ATP.

Why does lowering activation energy speed a reaction?

More reactant molecules can reach the transition state per unit time.

What is lysozyme?

A polysaccharide hydrolase that cleaves bacterial cell-wall peptidoglycan.

Which bacteria are especially vulnerable to lysozyme in the notes?

Gram-positive bacteria, whose wall contains exposed peptidoglycan.

What is peptidoglycan?

A bacterial cell-wall mesh of sugar chains cross-linked by short peptides that gives strength and support.

What are the 2 peptidoglycan sugars?

N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

What bond joins neighboring NAG and NAM sugars in the figure?

A β(1→4) glycosidic bond.

What bond does lysozyme hydrolyze?

The β(1→4) glycosidic bond between NAG and NAM in peptidoglycan.

What residues are central to lysozyme's catalytic mechanism?

Glu35 and Asp52.

What does Glu35 do in the lysozyme mechanism?

It initially donates a proton to the glycosidic oxygen and later helps activate water for hydrolysis.

What does Asp52 do in the lysozyme mechanism shown?

It stabilizes the cation-like intermediate and can form a covalent glycosyl-enzyme intermediate.

What happens to the bound sugar chain in the lysozyme active site?

One sugar is distorted toward a less stable conformation that favors cleavage.

Why is Glu35 able to retain a proton in the lysozyme active site?

Its relatively dry, nonpolar environment favors the protonated state.

Why can Asp52 act as a nucleophile in the mechanism?

Its more polar environment favors a negatively charged carboxylate that attacks the anomeric carbon.

What happens after water reacts in the lysozyme cycle?

The glycosyl-enzyme intermediate is hydrolyzed and sugar products are released.

What do E, S, ES, EP, and P mean in an enzyme mechanism?

E = enzyme; S = substrate; ES = enzyme–substrate complex; EP = enzyme–product complex; P = product.

What is the sequence of the simplified enzyme cycle?

E + S ⇌ ES → EP → E + P.

What happens to the catalyst after an enzyme-catalyzed reaction?

It is regenerated rather than consumed.

What relationship do Michaelis–Menten kinetics describe?

How initial enzyme reaction rate depends on substrate concentration.

What is Vmax?

The limiting maximum initial rate(velocity) reached as enzyme active sites become saturated with substrate.

What happens to rate as substrate concentration becomes very high?

The rate approaches Vmax and becomes less sensitive to further increases in substrate.

What is Km by definition?

The substrate concentration at which the initial reaction rate equals Vmax/2; measure of binding affinity

How is Km found on a Michaelis–Menten plot?

Find Vmax/2 on the rate axis, move across to the curve, then down to the substrate-concentration axis.

Does Km always equal a binding dissociation constant?

No. A lower Km often suggests greater apparent substrate affinity, but Km also depends on catalytic rate constants.

What is the Michaelis–Menten equation?

v₀ = Vmax[S]/(Km + [S]).

Why can Vmax and Km be hard to read precisely from a hyperbolic plot?

The curve approaches Vmax asymptotically, so the plateau can be difficult to locate.

What is a Lineweaver–Burk plot?

A double-reciprocal plot of 1/v₀ versus 1/[S] that linearizes Michaelis–Menten data; used to determine Km and Max

What are the Lineweaver–Burk intercepts?

y-intercept = 1/Vmax; x-intercept = −1/Km; slope = Km/Vmax.

What is a cofactor?

A nonprotein component, such as a metal ion or organic molecule, needed for or assisting protein function.

What is a prosthetic group?

A nonprotein cofactor tightly bound to a protein and needed for its function.

Why do cofactors extend an enzyme's capabilities?

They supply chemical properties or reaction chemistry that amino acid side chains alone may lack.

What is Heme, contains iron (Fe)?

A prosthetic-group example

What is allostery?

Binding or a change at one site alters a protein's shape or activity at another site.

What is an allosteric site?

A regulatory binding site distinct from the active site.

What is allosteric regulation?

Occurs when a regulator binds to an allosteric site (distinct from active site) causing/stabilizing a conformational change that alters protein activity

What is positive allosteric regulation?

An activator binds and favors an active conformation, increasing activity.

What is negative allosteric regulation?

An inhibitor binds and favors an inactive conformation, reducing activity.

What is cooperative binding in hemoglobin?

Oxygen binding to one subunit increases the oxygen affinity of other subunits.

How many subunits does hemoglobin have?

Four.

What is the shape of hemoglobin's cooperative oxygen-binding curve?

Sigmoidal (S-shaped).

What does oxygen binding do to hemoglobin's conformation?

It favors the higher-affinity conformation and facilitates oxygen binding by other subunits.

What is feedback or end-product inhibition?

The end product of a pathway inhibits an earlier step to limit its own overproduction.

What is ATCase?

Aspartate carbamoyltransferase, an allosteric enzyme in pyrimidine biosynthesis.

What product inhibits ATCase?

CTP, a pyrimidine-pathway end product.

Where does CTP bind ATCase?

At an allosteric regulatory site rather than the catalytic site.

What does CTP binding do to ATCase?

Stabilizes a less active conformation and decreases pathway activity and CTP production.

What is reversible phosphorylation?

Protein function regulation by adding and removing a phosphate group from a protein.

Why is phosphorylation called a covalent modification?

The added phosphate is covalently attached to the protein.

What determines a phosphoprotein's phosphorylation state?

The competing activities of protein kinases and protein phosphatases.

Where does a protein kinase typically obtain its phosphate?

ATP.

Can phosphorylation both activate and inhibit proteins?

Yes; its effect depends on the protein and site, often through a conformational change.

What happens if phosphatase activity predominates over kinase activity?

The protein tends toward a dephosphorylated state.

What are the two reversible protein switches emphasized in the notes?

Phosphorylation/dephosphorylation and GTP/GDP binding controlled by nucleotide exchange and hydrolysis.

What is a small GTPase or monomeric G protein?

A signaling protein that cycles between GTP-bound active and GDP-bound inactive states.

What nucleotide state usually turns a small GTPase on?

GTP-bound.

What nucleotide state usually turns a small GTPase off?

GDP-bound.

What event turns off a GTP-bound GTPase?

Hydrolysis of bound GTP to GDP.

What does a GAP do?

A GTPase-activating protein accelerates GTP hydrolysis, promoting the off state.

What does a GEF do?

A guanine nucleotide exchange factor promotes GDP release so GTP can bind, promoting the on state.

How do GAPs and GEFs regulate a small G protein?

They shift the balance between GDP-bound off and GTP-bound on forms.

What is EF-Tu?

A GTPase elongation factor that delivers aminoacyl-tRNA to the ribosome's A site.

What triggers EF-Tu's GTP hydrolysis?

Correct codon–anticodon recognition by the delivered tRNA.

What follows GTP hydrolysis by EF-Tu?

A conformational change releases the aminoacyl-tRNA for ribosomal accommodation.

What is regulation by subunit interaction?

Association of protein components (subunits) changes a protein's activity/function or target specificity.

What does CDK stand for?

Cyclin-dependent kinase.

What does cyclin binding do to a CDK?

Helps activate the kinase and influences which substrates it phosphorylates.

What is substrate specificity?

Which substrate molecules or proteins an enzyme recognizes and acts upon.

Why are different cyclins made at different cell-cycle times?

They activate particular CDKs and direct phosphorylation of relevant targets at appropriate times.