Kinetics Of Chymotrypsin Catalysis

2319 Words 10 Pages
Sarkis Hamalian
To examine the enzyme kinetics of chymotrypsin during the catalysis of the synthetic substrate p-nitrophenyl acetate (NPA). Initially, observe the magnitude of the initial burst with regards to the enzyme concentration. Secondly, the analysis of the steady state deacylation rate with regards to the pH of the reaction mixture. Chymotrypsin catalysis is a process that proceeds with two steps, an initial fast step involving the covalent modification of the enzyme to the substrate and the subsequent slower deacylation step which returns the enzyme to its free state (rate determining step). These steps are highly dependent on enzyme concentration and the pH of the environment in which the reaction
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The focus of this experiment will be chymotrypsin which falls under the category of serine proteases. Chymotrypsin has been discovered as a digestive enzyme found in pancreatic secretions of animals, whose main function is said to be hydrolysis of peptides that enter the duodenum of the small intestine. As can be determined from its enzyme class, it has an active serine residue which is used to hydrolyze aromatic amino acids such as Phenylalanine, Tyrosine or Tryptophan at their C-terminus. The hydrophobic property of the enzyme pocket (1 per enzyme) is what attracts the aromatic amino acids for cleavage. The catalytic strategies used by chymotrypsin are covalent and acid-base catalysis which are supported by the catalytic triad of serine (covalent catalysis), Histidine and Asparagine (acid-base catalysis). The chymotrypsin catalysis is carried out through two steps the first and faster step being the nucleophilic attack of the carbonyl group at the peptide bond by the serine residue resulting in a covalent modification. The stabilized enzyme intermediate undergoes the slow but essential second step in which water is used to release the covalent bond and regenerate the enzyme. In this case, burst kinetics is examined where the rate limiting step occurs …show more content…
In this case, we were able to prove that chymotrypsin works ideally at pH range between 7 and 8. As can be seen from graph 3 the rate clearly undergoes a rapid rise around pH 7 and continues to the max at pH 8. This increase in reaction rate can be accredited to the specific qualities of the acyl-enzyme intermediate and the reaction environment required to allow the steady state deacylation to proceed. This can be explained by examining the catalytic triad of chymotrypsin, mentioned earlier, and its importance in the activity of the enzyme. In particular we can examine the histidine residue of the triad, which in the second step is responsible for being protonated by water which ultimately results in the release of acetate from the serine residue resulting in a free enzyme that is able to react with a new substrate. The significance lies in the pKa of the Histidine side chain which is 6.8. So when the pH of the reaction mixture is above this pKa, ideally pH 7-8, the side chain can be protonated to allow the steady state deacylation step to occur

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