enzyme kinetics biochem

What do enzymes change about a reaction?

Lower activation energy and increase reaction rate without changing equilibrium.

What is the difference between an apoenzyme and holoenzyme?

Apoenzyme lacks its helper molecule (Prosthetic group); holoenzyme includes its helper molecule (Prosthetic Group)

What does negative ΔG mean?

Spontaneous and exergonic reaction.

What does positive ΔG mean?

Nonspontaneous and endergonic reaction.

What does zero ΔG mean?

The reaction is at equilibrium.

How do lock-and-key and induced-fit models differ?

Lock-and-key has a preexisting fit; induced fit changes the active site's shape.

What is the Michaelis-Menten equation?

v₀ = Vmax[S] / (Km + [S])

What does Km represent?

Substrate concentration at half of Vmax.

What does a low Km generally indicate?

Higher substrate affinity.

What is kcat?

Turnover number; kcat = Vmax / total enzyme concentration.

What happens to velocity at very high substrate concentrations?

Velocity approaches Vmax as the enzyme becomes saturated.

How does competitive inhibition affect Km and Vmax?

Km increases; Vmax stays the same.

How does uncompetitive inhibition affect Km and Vmax?

Both Km and Vmax decrease.

How does pure noncompetitive inhibition affect Km and Vmax?

Km stays the same; Vmax decreases.

How do sequential and ping-pong reactions differ?

Sequential binds all substrates before product release; ping-pong releases a product before all substrates bind.

What is an enzyme?

A protein or RNA molecule that catalyzes a biochemical reaction.

How are most enzymes commonly named?

Substrate + reaction type plus the suffix -ase.

What does an EC number classify?

An enzyme by the type of reaction it catalyzes.

What does EC 1 oxidoreductase do?

Catalyzes oxidation-reduction reactions. (Lactate/Dehydrogenase)

What does EC 2 transferase do?

Transfers chemical groups (C, N, P) between molecules. (Kinase)

What does EC 3 hydrolase do?

Breaks bonds by adding water. (Urease)

What does EC 4 lyase do?

Cleaves certain bonds (C-C, C-S, and C-N) without hydrolysis or oxidation. (Aldolase)

What does EC 5 isomerase do?

Catalyzes racemization of optical or geometric isomers (rearranges atoms within a molecule without changing number of atoms)

Ex: Mutase, Isomerase

What does EC 6 ligase do?

Catalyzes formation of bonds (btwn C, O, S, N) coupled to/ using energy from high-energy phosphate hydrolysis.

What does EC 7 translocase do?

Moves molecules across a membrane (One side to the other side)

What does a kinase do?

Transfers a phosphate group from one molecule (usually ATP) to another molecule.

How is a mutase different from a kinase?

A mutase moves a phosphate group within one molecule; a kinase transfers phosphate between molecules.

What does a dehydrogenase do?

Catalyzes redox reactions often using NADH or FADH.

What does aldolase do?

Cleaves a carbon-carbon bond in the reverse of aldol condensation.

What is an enzyme active site?

A small three-dimensional pocket created by the R groups of amino acids that binds substrate and performs catalysis.

*brought together by folding of protein into a 3D structure

What is a cofactor?

A dissociable (from enzyme) metal ion that helps enzyme activity such as Mg2+ or Fe2+.

What is a coenzyme?

A dissociable organic helper molecule such as NADH, FADH, NADHP

What is a prosthetic group?

An organic (nonprotein) helper group tightly or covalently attached to an enzyme.

What is entropy?

A measure of disorder or randomness.

What is Gibbs free-energy change at constant temperature?

Difference between reactants and products. (is rxn spon, nonspon, at equil)

(H, enthalpy) Total energy = (G, Gibbs) Usable energy + (S, entropy x T, temp K) unusable energy

G=H-TS

T (298 K)

Does a spontaneous reaction have to be fast?

No. -G predicts favorability and not reaction speed.

Does a catalyst alter reaction G?

No. Reaction ΔG is independent of the catalytic pathway.

Do enzymes shift the position of equilibrium?

No. They make equilibrium occur faster.

Energy position is a function only of the Free energy difference between reactants and products

How do enzymes accelerate a reaction?

They lower the activation energy by stabilizing the transition state.

How does protein folding help create an active site?

It brings distant amino-acid side chains together.

What do amino acids outside the active site (the ones that do not bind substrate) contribute?

A structural scaffold that “hold” the active site residues in the proper location

What microenvironment can an enzyme active site create?

A specialized nonpolar environment that may exclude water

Polar amino acid R groups acquire special properties

Which weak forces commonly stabilize enzyme-substrate binding?

Hydrogen bonds and van der Waals forces and hydrophobic interactions.

What is binding energy in enzyme catalysis?

Free energy released when enzyme binds its ligand (substrate)

What is first-order reaction kinetics?

Rate is proportional to one reactant concentration.

V= k [A]

What is second-order reaction kinetics?

Rate is proportional to the product of two concentrations or one concentration squared.

V=k[A]² or V=k[A][B]

What is a pseudo-first-order reaction?

If B is in excess concentration and A is low concentration, the reaction will be first order for A

What is zero-order reaction kinetics?

Rate is independent of reactant concentration.

Ex: Radioactive decay breaks down same rate regardless of concentration

Does changing enzyme concentration change Km?

No. Km is characteristic of an enzyme-substrate pair.

When does low Km imply stronger substrate binding?

When ES dissociation is much faster than conversion to product.

How does enzyme concentration affect Vmax?

Vmax increases in direct proportion to total enzyme concentration.

What happens when [S] equals Km?

v0 = Vmax / 2.

What does Vmax represent?

Maximum reaction velocity at enzyme saturation.

How does high Km affect substrate requirements?

More substrate is needed to approach enzyme saturation.

What happens to enzyme activity at excessively high temperature?

Activity falls as the enzyme denatures.

Why does pH affect enzyme activity?

It changes ionization of catalytic amino-acid side chains.

What does catalytic efficiency measure?

kcat/Km compares effective catalytic performance for different substrates.

What is a Lineweaver-Burk plot?

A double-reciprocal plot of 1/v0 versus 1/[S].

What is the Lineweaver-Burk y-intercept?

1 / Vmax.

What is the Lineweaver-Burk x-intercept?

-1 / Km.

What is the Lineweaver-Burk slope?

Km / Vmax.

What characterizes reversible inhibition?

Inhibitor binding is weak and enzyme activity can recover on dilution.

What characterizes irreversible inhibition?

Tight or covalent inhibition that does not recover readily on dilution.

What are two examples of irreversible enzyme-inhibiting drugs?

Penicillin inhibits transpeptidase; aspirin modifies cyclooxygenase.

Where does a competitive inhibitor bind?

At the substrate-binding active site.

Can increasing substrate overcome competitive inhibition?

Yes. More substrate can outcompete inhibitor.

What is the Lineweaver-Burk pattern for competitive inhibition?

Lines intersect on the y-axis.

What enzyme do statins competitively inhibit?

HMG-CoA reductase in cholesterol biosynthesis.

Where does an uncompetitive inhibitor bind?

Only to the enzyme-substrate complex.

Can increased substrate overcome uncompetitive inhibition?

No. Both apparent Km and Vmax decrease.

What is the Lineweaver-Burk pattern for uncompetitive inhibition?

Parallel lines with the same slope.

Where does a pure noncompetitive inhibitor bind?

An allosteric site on free enzyme or ES with equal affinity.

Can increased substrate overcome pure noncompetitive inhibition?

No. Vmax decreases even at high substrate.

What is the Lineweaver-Burk pattern for pure noncompetitive inhibition?

Lines intersect on the x-axis.

What is a sequential multisubstrate reaction?

Every substrate binds before any product is released.

What is an ordered sequential reaction?

Substrates bind and products leave in a required sequence.

What is a random sequential reaction?

Substrates may bind in either order before products are release