Kinetic investigation of the alpha-chymotrypsin-catalyzed hydrolysis of peptide substrates. The relationship between the peptide structure C-terminal to the cleaved bond and reactivity.
Peptide substrates of the general structure Ac-Tyr-Ly1-Ly2-..-Lym-NH2 and Ac-Phe-Ly1-NH2 have been synthesized and subjected to alpha-chymotrypsin-catalyzed hydrolysis to collect information on the interactions between the enzyme active site and the amino-acid residues Ly1, Ly2, etc., C-terminal to the susceptible bond of the peptide. For this purpose changes in the dissociation constants of the enzyme-substrate complexes and in the rate constants of acylation have been related to the structural variations of the substrates. The results indicate that interactions occur with the two residues next to the scissible bond, Ly1 and Ly2, but not with residue Ly3. Structural description of individual interactions was carried out with the aid of skeletal models of the active site. From such combination of kinetic and structural data a plausible interaction scheme for the substrate side C-terminal to the scissible bond has been deduced. This interaction scheme, which defines conformation and orientation of this part of the substrate within the active site, is characterized by the presence of a single hydrogen bond occurring between NH(Ly2) and O(Phe-41). No donor interacting with the back-bone carbonyl groups of residues Ly1 and Ly2 could be detected in the model of alpha-chymotrypsin. The effect of modification of the side chains of residues Ly1 and Ly2 on the kinetic constants was shown to be consistent with the interactions assumed to occur between the side chains of these residues and the active site. The interpretation of the results obtained from these specificity studies have led to refined concepts concerning the relative importance of different sets of enzyme-substrate interactions in determining reactivity.
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000378:metabolism*; D013329:Structure-Activity Relationship
D009842:Oligopeptides; D002918:Chymotrypsin