enzyme mechanisms

What are the catalytic strategies listed in the mechanism slides?

Acid-base catalysis, covalent catalysis, metal-ion catalysis, and proximity/orientation effects.

What is acid-base catalysis?

Catalysis by proton donation or acceptance.

What is general acid-base catalysis?

Proton transfer involving catalytic groups other than just solvent H3O+ or OH-.

What is specific acid-base catalysis?

Catalysis by hydronium or hydroxide from solvent.

What is covalent catalysis?

Formation of temporary covalent enzyme-substrate intermediate.

What is metal-ion catalysis?

A metal ion stabilizes charges, orients reactants, or assists chemical transformation.

What is the metal-ion example in the slides?

Zn2+ in carbonic anhydrase.

What does Zn2+ act as in carbonic anhydrase? [SLIDES]

Electrophilic metal center that helps activate bound water.

What is proximity/orientation catalysis?

Correct positioning and close proximity of reactants increases reaction rate.

What is the imidazole proximity example? [SLIDES]

Covalently attached imidazole made an intramolecular reaction 24 times faster than free imidazole in solution.

Why did the attached imidazole react faster? [SLIDES]

Both proximity and favorable orientation.

What is a nucleophile?

Electron-pair donor that attacks an electrophilic center.

What is an electrophile?

Electron-pair acceptor attacked by a nucleophile.

Which geometric isomers are shown on mechanism slides?

Maleate and fumarate.

What is maleate relative to fumarate?

Cis/trans (geometric) diastereomers, not enantiomers.

Which is cis: maleate or fumarate?

Maleate is cis; fumarate is trans.

What is the new seventh enzyme class mentioned in slides?

Translocases.

What do translocases do? [SLIDES]

Move molecules across or within membranes.

What is chymotrypsin?

Serine protease that hydrolyzes peptide bonds.

Which side of a residue does chymotrypsin cleave? [SLIDES]

Carboxyl side of large hydrophobic amino acids.

Why does chymotrypsin prefer hydrophobic side chains? [SLIDES]

Hydrophobic specificity pocket accommodates large hydrophobic side chains.

What are the three catalytic triad residues?

Ser195, His57, Asp102.

What does Ser195 do? [SLIDES]

Acts as nucleophile and forms temporary covalent bond with substrate.

What does His57 do?

Transfers protons during catalysis.

What does Asp102 do?

Helps orient and stabilize His57.

Why is the catalytic triad necessary? [SLIDES]

Peptide amide bonds are stable; the triad helps make active-site Ser a stronger nucleophile.

Which active-site residue is crucial for chymotrypsin nucleophilic attack?

Ser195.

What reagent modifies chymotrypsin active-site His? [SLIDES]

TPCK.

Why does TPCK bind chymotrypsin? [SLIDES]

It structurally resembles substrates and binds active site.

What does TPCK modification show?

Active-site His is important for catalysis.

What is the chymotrypsin mechanism called? [SLIDES]

Double displacement or ping-pong mechanism.

What intermediate explains ping-pong kinetics? [SLIDES]

Acyl-enzyme intermediate.

How many major stages are in chymotrypsin mechanism?

Two: acylation and deacylation.

What happens in acylation?

Ser attacks substrate; tetrahedral intermediate forms; first product leaves; acyl-enzyme remains.

What happens in deacylation?

Water attacks acyl-enzyme; tetrahedral intermediate forms; second product leaves; enzyme regenerates.

What happens during the first nucleophilic attack?

Ser195 oxygen attacks peptide carbonyl carbon.

What is the first tetrahedral intermediate?

Transient species with negatively charged carbonyl oxygen after Ser attack.

What stabilizes tetrahedral intermediates in chymotrypsin?

Oxyanion hole.

What is an oxyanion hole?

Active-site region that stabilizes negatively charged oxygen through hydrogen bonds.

What is released at end of acylation?

Amine-containing peptide fragment.

What remains after first product leaves?

Covalent acyl-enzyme intermediate.

What nucleophile attacks in deacylation?

Water activated by His57.

What is released at end of deacylation?

Carboxyl-containing peptide fragment.

What is regenerated after deacylation?

Free active enzyme.

What stage is rapid in the chymotrypsin kinetic scheme shown? [SLIDES]

Stage 1.

What kinetic feature supports the acyl-enzyme intermediate? [SLIDES]

Two-stage burst/ping-pong behavior.

What does the mechanism explain according to slides?

Kinetics and inhibition data.

What is the order of chymotrypsin key intermediates?

ES → tetrahedral intermediate → acyl-enzyme → tetrahedral intermediate → E + products.

Which catalysis strategies operate in chymotrypsin?

General acid-base and covalent catalysis, with transition-state stabilization.

What is the key acid-base residue in chymotrypsin?

His57.

What is the key covalent-catalysis residue in chymotrypsin?

Ser195.

What is the key stabilization feature of chymotrypsin?

Oxyanion hole stabilizes tetrahedral intermediate.

What is the highest-yield chymotrypsin memory line?

Ser attacks; His transfers H+; Asp stabilizes His; acyl-enzyme forms; water regenerates enzyme.