USE OF SPECIFICITY CONSTANT OF ALPHA-CHYMOTRYPSIN

The turnover constant, kcat, the Michaelis-Menten constant, Km, and the specificity constant, Kcat/Km, were calculated for the α-chymotrypsin-catalyzed hydrolysis of the N-3-(2-furyl)acryloylamides of L-tryptophan, L-phenylalanine, L-α-aminoheptanoic acid, and L-leucine. No correlation was observed between the structure (hydrocarbon side-chain character) of these amino acid derivatives and either kcat or Km, but an excellent correlation was found between the structure and the specificity constant, kcat/Km. The relative specificities kcat1/Km1/ kcat2/Km2 between any of the above amides was the same (within experimental error) as those calculated previously between the analogous N-acetylamino acid methyl esters. Therefore, the proposal that the specificity constant is the most accurate reflection (of all kinetic constants) of the α-chymotrypsin specificity for a substrate is confirmed. From this proposal (1) an interpretation is established for the binding specificity of a substrate to the enzyme, and (2) the specificity constant and relative specificity can be used to predict the behavior of α-chymotrypsin toward any given substrate which is a derivative of an α-L-amino acid. © 1969, American Chemical Society. All rights reserved.