Open AccessKinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates
Author(s) -
Baumann Werner K.,
Bizzozero Spartaco A.,
Dutler Hans
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb03136.x
Subject(s) - chemistry , active site , peptide bond , stereochemistry , peptide , substrate (aquarium) , hydrogen bond , hydrolysis , residue (chemistry) , side chain , dissociation constant , amino acid , crystallography , enzyme , molecule , organic chemistry , biochemistry , biology , ecology , receptor , polymer
A number of peptide substrates of the general structure Ac‐L xn ‐ … ‐L x 2 ‐L x 1 ‐Gly‐NH 2 have been synthesized and their α‐chymotrypsin‐catalyzed hydrolyses studied. The acylation rate constants, k 23 (= k cat ), and the dissociation constants of the enzyme‐substrate complexes, K EA (= K m ), have been determined using a modified pH‐stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N‐terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme‐substrate interaction scheme proposed in 1971 by Segal et al . ( Biochemistry 10 , 3728). A comparison of the k 23 / K EA values indicates that the influence of a single structural change on the overall reactivity is virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k 23 and K EA values shows that, in general, changes in substrate structure are mainly reflected by changes in k 23 rather than in K EA . A few exceptions have been found: K EA or K EA and k 23 change when glycine is introduced at the x 2 position, when this glycine is replaced by alanine or when alanine is introduced at the x 3 position.