Unit 20 - Examples of Enzyme Reactions

What type of catalysis does RNase A use?

General acid-base catalysis.

What reaction does RNase A catalyze?

Hydrolysis of RNA to 2’-3’ cyclic nucleotide intermediates.

Which residues are critical for RNase A activity?

Histidine 12 and Histidine 119.

Why are histidine residues effective in RNase A?

Their pKa (~6) allows them to act as acid/base near physiological pH.

What is the role of His12 in RNase A?

Acts as a general base in the first step and a general acid in the second.

What is the role of His119 in RNase A?

Acts as a general acid in the first step and a general base in the second.

What is the kcat of RNase A?

Approximately 323 s⁻¹.

What enzyme uses Zn²⁺ to catalyze reactions?

Carbonic anhydrase.

What reaction is catalyzed by carbonic anhydrase?

CO₂ + H₂O ⇌ HCO₃⁻ + H⁺.

What residue coordinates the Zn²⁺ in carbonic anhydrase?

Histidine.

What role does His64 play in carbonic anhydrase?

It shuttles protons in and out of the active site.

Why is Zn²⁺ important in carbonic anhydrase?

It lowers the pKa of water to generate OH⁻ for nucleophilic attack.

What is the general mechanism of carbonic anhydrase?

Zn²⁺-activated water attacks CO₂ to form bicarbonate.

What is the typical structure of the carbonic anhydrase active site?

A Zn²⁺ coordinated by 3 His residues and a water.

What type of catalysis does chymotrypsin use?

Covalent catalysis and acid-base catalysis.

What type of bonds does chymotrypsin cleave?

Peptide bonds, specifically near aromatic residues.

What artificial substrate is used to assay chymotrypsin?

Para-nitrophenyl acetate.

What observation supports the two-step mechanism of chymotrypsin?

Initial-burst kinetics indicating an acyl-enzyme intermediate.

What reagent irreversibly inhibits chymotrypsin?

Diisopropylfluorophosphate (DIFP).

Which residue does DIFP modify in chymotrypsin?

Serine in the active site (Ser195).

What are the three key residues in the chymotrypsin catalytic triad?

Asp102, His57, Ser195.

What is the function of the catalytic triad in chymotrypsin?

To polarize Ser195 and enhance its nucleophilicity.

What stabilizes the transition state in chymotrypsin?

The oxyanion hole.

What is the magnitude of rate enhancement by chymotrypsin?

~10¹⁰-fold over uncatalyzed hydrolysis.

What structural model mimics the transition state of chymotrypsin?

Trypsin-BPTI complex.

What is BPTI?

Bovine pancreatic trypsin inhibitor that forms a tight complex with trypsin.

What does the BPTI structure reveal?

A trapped tetrahedral intermediate-like state.

What determines substrate specificity in serine proteases?

The shape and properties of the substrate binding pocket.

What residues do chymotrypsin, trypsin, and elastase prefer?

Aromatic, basic, and small residues, respectively.

What is the catalytic mechanism of hexokinase?

Phosphorylation of glucose using ATP.

What conformational change occurs in hexokinase?

Domain movement that closes around glucose and ATP.