Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide‐Ester Substrates

Abstract

A number of peptide-ester substrates of the general structure Ac-L,,-. . .-L,,-L,,-OMe have been synthesized and their a-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-L,~-L,,-OM~ and the amino-acid derivatives Ac-L,,-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, k3,. For the tripeptide ester Ac-Ala-Ala-Tyr-OMe it appears that the rate constant for the dissociation of the enzyme-substrate complex, kZ1, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k , , and k2, could be determined and the values of k23, k32 and k3, 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 k2JKEA, increases 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 k,, and on KEA is dependent on the nature of the other interactions. However, the effect on 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-L,,-. . .-Lx2-. . .-L,,-Gly-NH2 which have the same acyl moiety as the peptide esters studied in this work.The interactions of peptide substrates with the active site of a-chymotrypsin are known to involve several amino-acid residues on both sides of the bond to be cleaved. The most important interactions for determining the reactivity of a substrate are due to the residue containing the carbonyl group of the cleaved bond (primary interactions), but recent evidence indicates that interactions involving adjacent residues (secondary interactions) also have a significant influence on reactivity. Structural information about secondary interactions between the enzyme and the residues on the acyl side of peptide substrates has been provided by a crystallographic study of y-chymotrypsin inhibited by peptidyl chloromethyl ketones [l]. It was shown that the peptide moiety of the inhibitor forms an antiparallel 8-structure with the residues Ser-214, Trp-215 and Gly-216 of the enzyme.Abbreviations. IUPAC/IUB rules for peptides are followed, Enzyme. a-Chymotrypsin (EC 3.4.21.1).see Eur. J . Biockern. (1972) 27, 201 -207. On the basis of this work an interaction scheme had been proposed whose general validity for substrates [2,3] and non-covalent...

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

“…Data existing in the literature for Z-Xaa-Lys-OMe substrates of trypsin [56], where k,,, follows the same order as in the present Ac-Xaa-Arg-OMe series, as well as Ac-Xaa-Tyr-OMe substrates of chymotrypsin [47] also allow this effect to be recognized. Kunugi et al [48] suggested the enforcement of an unfavourable position of the acylenzyme ester bond relative to His-57 by a tightly binding Pz residue as explanation for the lower k 3 observed for such a chymotrypsin substrate.…”

“…This value is about fourfold higher than that presently determined for the corresponding methyl esters, though a really valid comparison is hampered by differences in reaction conditions. The behaviour of tissue kallikrein is quite different from that of chymotrypsin, where it has been shown that variation of the P2 residue influences k,,,lK, to a similar extent with both acetyl dipeptide glycinamides and esters [47]. In the tissue-kallikrein-catalyzed reactions, there might exist influences from the different binding modes or intrinsic reactivities of the various leaving groups.…”

Esterification of n-protected-tyrosine by alpha-chymotrypsin in a high concentration of ethanol

“…According to Bizzozero et al (1975), the S 2 subsite of alpha-chymotrypsin is formed by His-57 and Ile-99 carbon side chains and by the alpha and beta carbons of Trp-215, which favor interaction with the side chains of hydrophobic amino acids. Therefore, the esterification rates of Boc-Tyr-OH and Z-Tyr-OH, whose protective groups are hydrophobic, would be expected to be higher than that of Ac-Tyr-OH.…”

Serine‐Protease‐Assisted Synthesis of Peptide Substrates for α‐Chymotrypsin

“…To a solution of 315 mg (1.61 mmol) 2y2) in 2.5 ml H20, adjusted to pH 8.6 with 0.4 ml of 2~ NaOH, 307 mg (1.04 mmol) 2x [20] dissolved in 3 ml MeOH were added. The reaction was started by the addition of 100 pI of a 15 p~ 6-chymotrypsin.…”