Chymotrypsin active center amino substrate specificity

Active center that part of an enzyme or other protein which binds the specific substrate and converts it to product or otherwise interacts with it. The A.C. thus consists of the actual catalytic center, which is relatively unspedfic, and the substrate-binding site, which is responsible for the specificity of the enzyme. Usually only a few amino acid residues interact directly with the substrate in the A.c. the rest of the protein molecule serves to hold these few in the proper configuration. The amino acids involved in catalysis may lie at a considerable distance from each other in the absence of substrate they are brought into play by conformational changes induced by substrate binding (see Cooperativity model, Chymotrypsin, Serine proteases). 

A second elastolytic enzyme (Af, 21,900) has been isolated from porcine pancreas. It shows higher activity than chymotrypsin in the hydrolysis of acetyltyrosine ester, which is used routinely to assay chymotrypsin. Another E,-like enzyme, a-lytic proteinase (Af, 19,900, 198 amino acids) has been isolated from the soil bacterium Myxobacter 495. This enzyme is remarkably similar to pancreatic E. both in structure (41 % homology, sequence in the active center Gly-Asp-Ser-Gly, 3 homologous disulfide bridges) and substrate specificity. Another E. (Af, 22,300) has been isolated from Pseudomonas aeruginosa. 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

This biphenyl model compound was shown to possess the so-called primary optical specificity of a-chymotrypsin. That is, only the enantiomer related to L-phenylalanine was hydrolyzed by the enzyme. Generally speaking, there are three more levels of specific substrate recognition in enzyme catalysis. Let us consider a peptide bond in a polypeptide chain. The lateral side chain R2 is responsible for the normal specificity of the enzyme. For a-chymotrypsin, R2 is an aromatic side chain and the hydrophobic cavity a aromatic hole in the active center is there to accommodate the amino acid to be recognized by the enzyme. This is referred to as the primary structural specificity. 

In order to verify this assumption, we studied the temperature dependence of the kinetic isotope effect during hydrolysis of some specific substrates, i.e. amino acid esters, by serine proteases like a-chymotrypsin[468,469] and 3-trypsin[470]. It is well known that a two-stage hydrolysis mechanism takes place for this class of enzymes with the formation, as an intermediate product, of an acyl derivative of the amino acid serine (Ser-195), a constituent of the polypeptide chain of the protein. The side group (-CH2OH) of serine is located in the region of the active center[471-474] ... 

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