The kinetics of hydrolysis of some extended N-aminoacyl-L-phenylalanine methyl esters by bovine chymotrypsin A-alpha. Evidence for enzyme subsite S5.
A series of N-acetylated peptide methyl esters of general formula N-acetyl-(glycyl)n-L-phenylalanine methyl ester (n = 0--3) has been synthesized to study the effect of varying aminoacyl chain length on the efficiency of chymotrypsin A alpha (EC 3.4.21.1) catalysed ester hydrolysis. Values of kcat and Km for each substrate have been obtained from kinetic measurements at pH 8.00 and 25.0 degrees C. It has been found that for the first three members of the series (n = 0--2) there is an increase in kcat value as the aminoacyl chain length is increased. However, the kinetic constants (kcat and Km) for the third (n = 2) and fourth (n = 3) members of the series were found to be very similar. These results are shown to be consistent with a substrate binding scheme proposed for the isomeric enzyme chymotrypsin A gamma. The enzyme-catalysed reactions were also investigated over a range of temperature (15--35 degrees C). In each case the Arrhenius law was obeyed, within experimental error, and evaluation of meaningful values for the thermodynamic functions of activation (delta H++ and delta S++) was possible with certain assumptions. In contrast to the similarly of kinetic constants found for the third and fourth members of the substrate series, the corresponding values of delta H++ and delta S++ were markedly different. These results, together with those for the first two members of the series are interpreted in terms of a model binding system which is consistent with the existence of further enzyme subsites in the S4--S5 region.
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D010649:Phenylalanine; D002918:Chymotrypsin; D014357:Trypsin