Bio 40 Lec 11 Catalysis

What are the two components of enzymatic catalysis, and what overall rate acceleration do they produce?

Binding (E+S to ES) and chemistry (ES to EP); together they give 10^7 to 10^14-fold rate acceleration.

Contrast the 'lock and key' model with the modern theory of active-site complementarity.

Lock and key: active site complementary to the substrate. Modern theory: active site complementary to the transition state.

Why would an active site that's perfectly complementary to the substrate (strict lock-and-key) actually work against catalysis?

It would stabilize the ground-state substrate, which works against the reaction proceeding toward the transition state.

What is the quantitative relationship between activation energy decrease and rate increase, and how much energy can weak binding interactions contribute?

Every 5.7 kJ/mol decrease in activation energy gives a 10-fold rate increase; single interactions give 4-30 kJ/mol, multiple interactions sum to 60-100 kJ/mol.

At what point along the reaction coordinate is binding energy maximal, and why does this matter for catalysis?

The transition state -- which is why binding energy can be used to accelerate the reaction rather than just stabilizing the ES complex.

Give the approximate rate-increase contributions of proximity, orientation, and reduced bond motion (the three components of 'entropy reduction').

Proximity: ~5-fold. Orientation: ~100-fold. Reduced bond motion: ~10^7-fold.

Describe desolvation and strain as mechanisms by which binding energy overcomes reaction barriers.

Desolvation: enzyme replaces the substrate's H-bonds to water. Strain: enzyme distorts the substrate's geometry/electronics toward the transition state.

In the 'Stickase' thought experiment, what's the difference in outcome between an active site complementary to the substrate versus one complementary to the transition state?

Complementary to the substrate: stabilizes it, impeding bending/breaking. Complementary to the transition state: destabilizes the ground state, favoring the bent/high-energy shape and accelerating reaction.

What transition-state analog is used as evidence for transition-state binding in proline racemase, and how does its binding compare to the real substrate?

Pyrrole-2-carboxylate -- it binds proline racemase about 160x better than proline itself, as a competitive inhibitor.

What is a catalytic antibody (abzyme), and what does its existence demonstrate about binding energy and catalysis?

An antibody raised against a stable transition-state analog, which then catalyzes the real reaction using binding energy alone (no chemical catalysis); used for reactions lacking a natural enzyme.

Which amino acid side chains commonly serve as acids (proton donors) in general acid-base catalysis?

His, Cys, Ser, Glu/Asp, Lys/Arg.

Describe the roles of His12 and His119 in the RNase A catalytic mechanism, across both steps of the reaction.

His12: general base (removes proton, facilitates attack). His119: general acid (donates proton to leaving group). Roles flip when water hydrolyzes the intermediate.

Why does the RNase A mechanism resemble a ping-pong mechanism?

Water only participates after the first leaving group has already departed -- no ternary complex forms, just like a ping-pong (double displacement) mechanism.

How can you predict which residues are catalytically important just from an enzyme's pH-activity profile?

By looking at the pKa's (inflection points) where activity rises or falls in a pH-activity profile.

How does a nearby positive charge versus a nearby negative charge affect the pKa of a lysine side chain?

Nearby positive charge: decreases Lys pKa (easier to lose its proton). Nearby negative charge: increases Lys pKa (stabilizes the protonated form).

What is the defining mechanism of covalent catalysis, and what condition must the new pathway satisfy?

It forms a transient covalent bond between a catalytic group and the substrate, creating a new, lower-energy reaction pathway.

Define a nucleophile, and state how nucleophilicity relates to a group's pKa.

An electron-rich ion or molecule with a lone pair; nucleophilicity increases with increasing pKa.

How is covalent catalysis mechanistically related to general base catalysis?

Both use the same functional groups, but covalent catalysis forms a new covalent bond instead of just removing a proton.

In the chymotrypsin mechanism preview, describe how base catalysis and covalent catalysis work together involving Ser195.

Base catalysis first makes Ser195's oxygen more nucleophilic (deprotonation); then Ser195 performs covalent catalysis via nucleophilic attack on the substrate's carbonyl carbon, forming an acyl-enzyme intermediate.

Roughly what fraction of enzymes require a metal ion cofactor for activity?

About one third.

Give examples of tightly-bound versus loosely-bound metal cofactors in enzymes.

Tightly bound: Fe2+/Fe3+, Cu2+, Zn2+, Mn2+. Loosely bound: Na+, K+, Mg2+, Ca2+.

List the three main catalytic roles of metal ions in enzymes, and explain why metal ions are useful for charge stabilization compared to H+.

Orient substrates; mediate redox reactions via oxidation-state changes; provide electrostatic shielding of negative charges, which is useful since [H+] is very limited at pH 7.

Give the example used for 'catalysis by approximation' that also simultaneously illustrates metal ion catalysis.

Phosphoryl group transfer by nucleoside monophosphate kinases, where Mg2+ stabilizes ATP.

Why is 'catalysis by approximation' considered fundamentally a binding-energy mechanism rather than a new chemical mechanism?

It is fundamentally driven by binding energy (correct substrate orientation), the same underlying logic as entropy reduction, rather than a separate chemical mechanism.

If 100 units of enzyme are treated with an irreversible inhibitor that inactivates 50 of those units, what happens to the apparent Vmax, and why?

Only 50 units remain active, and since Vmax = kcat[Et], the apparent Vmax is cut in half.

Mechanistically, how does irreversible inhibition's effect on Vmax differ from a reversible inhibitor's effect on Vmax?

Reversible inhibitors act through binding equilibria with intact enzyme; irreversible inhibitors physically remove enzyme from the active pool altogether.