| Abstract: | A number of peptide-ester substrates of the general structure Ac-Lxn-...-Lx2-Lx1-OMe have been synthesized and their alpha-chymotrypsin-catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve. For the dipeptide esters Ac-Lx2-Lx1-OMe and the amino-acid derivatives Ac-Lx1-OMe the following constants could be determined: the dissociation constant of the enzyme-substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the deacylation step, k31. For the tripeptide ester Ac-Ala-Ala-Tyr-OMe it appears that the rate constant for the dissociation of the enzyme-substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increase with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect of k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac-Lxn-... Lx2-...-Lx1-Gly-NH2 which have the same acyl moiety as the peptide esters studied in this work. |
