9.9 Enzyme Mechanism and Catalysis II

Substrates can be aligned by binding to an enzyme so that

nucleophiles are position for reaction

An AA residue can function both as a

general acid and general base

Transition-State Analogs

Stable molecules designed to resemble transition states; type of irreversible inhibitors; binds more tightly than substrate

Protease catalyzes _______ _______ of ______ ____ at ___-terminal side

hydrolytic cleavage of peptide bonds; C-terminal

Chymotrypsin

bovine pancreatic protease that catalyzes the hydrolytic cleavage of aromatic AA peptide bond

Chymotrypsin cleaves polypeptide chains at _-terminal side of the aromatic amino acid residues:

C terminal; Phe, Trp, and Tyr

Chymotrypsin enhances rate by

10⁹

Phase 1 of hydrolysis reaction

Acylation of N-terminal with Phe

Phase 2 of hydrolysis reaction

Deacylation of N-terminal fragment (releasing Phe)

Hexokinase catalyzes the phosphorylation of

glucose in a reversible reaction, involving Mg ion, ADP and ATP

Phase 1 - Step 1, Chymotrypsin Reaction

Substrate binds to active site at Asp-102, His-57, and Ser-195, forming catalytic triad, Asp -102 makes His-57 a better general base in the first step

This step creates the ES complex

Phase 1 - Step 2, Chymotrypsin Reaction

His-57 acts as general base and deprotonates hydroxyl group on Ser-195 to make it a nucleophile.

Ser-195 then acts as a nuc to attack carbonyl group on substrate (Covalent catalysis)

Phase 1 - Step 3, Chymotrypsin Reaction

Short-lived protonated His-57 from step two acts as an acid and protonates the amine leaving group, causing the peptide bond to break and product 1 (Group on peptide bond) to leave

This step creates the acyl-enzyme intermediate

Phase 2 - Step 4, Chymotrypsin Reaction

Water enters site where product exited

Phase 2 - Step 5, Chymotrypsin Reaction

His-57 acts as a general acid to deprotonate water, making it more nucleophilic, the OH^- then attacks the carbonyl carbon

Phase 2 - Step 6, Chymotrypsin Reaction

Protonated His-57 acts as a general acid to protonate Ser-195 leaving group

Ser-195 cleaves from the N-term end of polypeptide (This regenerates the enzyme)

Phase 2 - Step 7, Chymotrypsin Reaction

The final product (N-term end of original substrate) leaves

Enzyme can restart reaction again

Explain pH dependence of chymotrypsin reactions; pH 7, 8, and 8.5

pH7: protonation of His57 stops enzyme function; pH8: optimal, His57 is unprotonated and Ile16 is protonated; pH8.5: Ile16 is deprotonated, stops enzyme function

Retrovirus

possesses an RNA genome and enzyme; a reverse transcriptase that uses RNA to direct synthesis of a complementary DNA; ex. HIV

HIV protease mechanism

Aspartyl protease: Utilizes water to cut amino acid leaving group; Water attacks carbonyl carbon to generate tetrahedral intermediate with Asp25, when it collapses, the amino group is expelled and protonated by another Asp as it leaves

HIV protease inhibitors

Irreversible inhibitors; forms a noncovalent complex with the enzyme; red hydroxyl groups acts as a transition-state analogs and blue benzyl groups targets binding site

Transition-state analogs

Inhibitors meant to mimic the high-energy, short-term intermediates of enzymes

Hexokinase induced fit; what causes hexokinase autophosphorylation?

Hexokinase phosphorylates glucose molecule; when Mg*ATP binds, the energy created results in a conformational change that makes hexokinase active and facilitate the phosphorylation; very specific to glucose. In presence of xylose, hexokinase is tricked to induce autophosphorylation.

Enolase; Enzyme requires what?; Provide mechanism

Catalyzes dehydration of 2-phosphoglycerate to phosphoenolpyruvate; Requires metal ions (Mg⁺²) which acts as a cofactor

Lys345 attracts a proton via base catalysis. 2 Mg⁺² stabilizes structure. Glu211 eliminates OH group by acid catalysis