Question

1.. Example questions to guide you through the mechanism study of Serine proteases

• Describe substrate binding, including the role and chemical nature of the "specificity pocket" in chymotrypsin, and which peptide bond in the substrate (relative to the specificity group) will be cleaved.
• Draw the structure of the catalytic triad at the beginning of the reaction, and explain how the states of ionization and hydrogen bonding pattern of those 3 groups change step by step during catalysis.
• Explain the role of each member of the catalytic triad in the reaction.
• What is the role of the "oxyanion hole" in the mechanism?
• Identify the nucleophile that attacks the carbonyl carbon in acylation;
• What would be the nucleophile that attacks the carbonyl carbon in deacylation?
• State what is being acylated and deacylated in the chymotrypsin 2 phases of reaction (be specific about the functional group involved).

Answer 1

Serine Protease:

Part-A: Describe substrate binding, including the role and chemical nature of the "specificity pocket" in chymotrypsin, and which peptide bond in the substrate (relative to the specificity group) will be cleaved.

The significant serine proteases geometry is enable them to recognize the substrate cleavage sites and they characteristically bind at the transition state, finally declines the overall activation energy of the reaction, and thereby enables the significant catalytic ability of the enzyme. These zymozens (serine proteases) are initially inactive in the pancreas. These are inactive precursors of the serine proteases synthesized in the pancreas, incase if they become active where they synthesized without recognizing the substrate site may leading to autolysis (self-digestion) of the enzyme. Further this self- digestion due to premature activation of serine proteases result in acute pancreatitis.

Substrate binding, including the role and chemical nature of the "specificity pocket" in chymotrypsin:

The crucial component in the mechanism of serine protease, chymotrypsin enzyme catalysis since it binds with the protein substrate to form a catalytic triad (His 57, ser 195 and aspar 102). This catalytic traid of chymotrypsin have the capability to recognize the catalytic sites of the substrate polypeptide as these substrates are in complex tertiary structure forms due to folding & it is referred as the specificity pocket.

His-57 aids in the nucleophilic attack by oxygen in Ser-195 by making H-bond at Schiff base nitrogen position of imidazole ring of histidine at step-2. Initially, during catalytic triad formation, it attracts proton from "OH" group of serine -195 and promote acyl-intermediate formation followed by nucleophillic attack on "carbonyl group of peptide bond". However, at step-6, it is not going to undergo "protonation" by Serine oxygen, conversely, serine-195 is going to accept proton from His-57 to restore original catalytic triad after "peptide bond" cleavage.

Part-B: Draw the structure of the catalytic triad at the beginning of the reaction, and explain how the states of ionization and hydrogen bonding pattern of those 3 groups change step by step during catalysis.

Chymotrypsin & oxyanion hole formation: Chymotrypsin is a serine protease and forms a tetrahydral intermediate with an oxyanion hole when digesting polyptide substrates,

Oxyanion hole -H-N H-N Tetrahedral intermediate Ser 195 Oxyanionhole in chymotrypsin His 57 H H Gly 193 NH Asp NH O-C-102 HN N R- H R Gly 193 zo

Oxyanion hole Tetrahedral intermediate Ser 195 Oxyanionhole in chymotrypsin His 57 0 У 1931 NH Gly 193

The carboxypeptidases are the proteolytic enzyme that cleaves carboxy side chain of the polypeptide substrate without forming any tetrahydral intermediate with “oxyanion hole” formation using its active sites. The oxyanion hole of chymotrypsin compare to that of carbopxypeptidase in having the following properties of catalytic activity.

The crucial component in the mechanism of serine protease, chymotrypsin enzyme catalysis as they bind with the substrate is the formation of catalytic triad (His 57, ser 195 and aspar 102). This catalytic traid of chymotrypsin have the capability to recognize the catalytic sites of the substrate polypeptide as these substrates are in complex tertiary structure forms due to folding.

His-57 aids in the nucleophilic attack by oxygen in Ser-195 by making H-bond at Schiff base nitrogen position of imidazole ring of histidine at step-2. Initially, during catalytic triad formation, it attracts proton from "OH" group of serine -195 and promote acyl-intermediate formation followed by nucleophillic attack on "carbonyl group of peptide bond". However, at step-6, it is not going to undergo "protonation" by Serine oxygen, conversely, serine-195 is going to accept proton from His-57 to restore original catalytic triad after "peptide bond" cleavage.

The significant serine proteases geometry is enable them to recognize the substrate cleavage sites and they characteristically bind at the transition state, finally declines the overall activation energy of the reaction, and thereby enables the significant catalytic ability of the enzyme.

Part-C: Explain the role of each member of the catalytic triad in the reaction.

The crucial component in the mechanism of serine protease, chymotrypsin enzyme catalysis as they bind with the substrate is the formation of catalytic triad (His 57, ser 195 and aspar 102). This catalytic traid of chymotrypsin have the capability to recognize the catalytic sites of the substrate polypeptide as these substrates are in complex tertiary structure forms due to folding.