Kinetic Investigation of the α-Chymotrypsin-Catalyzed Hydrolysis of Peptide Substrates.

A number of peptide substrates of the general structure Ac-Lxn … -Lx2-Lx1-Gly-N'H2, have been synthesized and their α-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, k23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, KEA (= Km), 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 k23/KEA 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 k23 and KEA values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: KEA or KEA and k28 change when glycine is introduced at the x2 position, when this glycine is replaced by alanine or when alanine is introduced at the x3 position. [ABSTRACT FROM AUTHOR]