Kinetic Investigation of the a‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Abstract

Peptide substrates of the general structure Ac-Tyr-Lyl-Lyz-. . -Ly,-NH2 and Ac-Phe-L,,-NHz have been synthesized and subjected to cr-chymotrypsin-catalyzed hydrolysis to collect information on the interactions between the enzyme active site and the amino-acid residues Lyl, Ly2, etc., C-terminal to the susceptible bond of the peptide. For this purpose changes in the dissociation constants of the enzyme-substrate complexes and in the rate constants of acylation have been related to the structural variations of the substrates. The results indicate that interactions occur with the two residues next to the scissible bond, L,1 and Lyz, but not with residue Ly3. Structural description of individual interactions was carried out with the aid of skeletal models of the active site. From such combination of kinetic and structural data a plausible interaction scheme for the substrate side C-terminal to the scissible bond has been deduced. This interaction scheme, which defines conformation and orientation of this part of the substrate within the active site, is characterized by the presence of a single hydrogen bond occurring between NH(Ly2) and . No donor interacting with the back-bone carbonyl groups of residues Lyl and LY2 could be detected in the model of a-chymotrypsin. The effect of modification of the side chains of residues Lyl and LY2 on the kinetic constants was shown to be consistent with the interactions assumed to occur between the side chains of these residues and the active site. The interpretation of the results obtained from these specificity studies have led to refined concepts concerning the relative importance of different sets of enzyme-substrate interactions in determining reactivity.Endopeptidases possess extended active sites which can accommodate between five and seven amino-acid residues of peptide substrates 11-51, Since they usually display a marked specificity for the side chain of the residue containing the reactive carbonyl group, the interactions of this residue with the enzyme are often termed primary interactions. For some serine proteases, as for trypsin, chymotrypsin and elastase, the structural basis of these interactions is clearly discernible from the known three-dimensional structures of the active sites. The other interacting residues of the substrate are said to form secondary interactions with the enzyme. On account of differences in the way these secondary interactions affect the rate of hydrolysis, they can be subdivided into two sets: those originating from the invariant part and those originating from the variable part of the peptide substrate. The invariant part includes the atoms of the backbone and the p carbons of the side chains, which are present in all amino acids but glycine. It is reasonable to assume that this invariant part, together with the specific residue, leads to interactions occurring according to a well defined interaction scheme which henceforth will be called the 'cardinal' interaction scheme. For chymotrypsin, such an interaction scheme regarding the substr...

The specificity of chymotrypsin

“…Further reduction of the number of S subsites diminishes the F value. This finding is in good agreement with the crystal structure of chymotrypsin [lo, 111 and model building analyses [12] which showed that the P3 residues of peptide substrates are involved in two hydrogen bonds with the enzyme. Table 1 shows that the contribution of the P2 and P3 fragments can also be deleted.…”

“…Table 1 shows that the contribution of the P2 and P3 fragments can also be deleted. However, we decided to include the P2 fragments in our calculations since the amide groups of these fragments are known to donate a hydrogen bond to the carbonyl oxygen of Phe41 of the enzyme [12]. There are two substrates in the data set with proline residues in the Pi position which, for structural reasons, can not form this hydrogen bond.…”

“…Crystal structure analyses [lo, 111 and model building [12] revealed that a number of H bonds are formed between the active site of the enzyme and peptide substrates. Sz subsite.…”

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

“…Remarkable are the outstanding substrate properties of Ac-Phe-Arg-Ser-Val-NH, for both enzymes. There might exist a specific interaction only possible with a C-terminal NH, group of the residue in Pz as proposed in the case of chymotrypsin [55].…”

Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

“…Pro are not preferred [27][28][29]. In addition to the three substrates used in this study, we synthesized Mca-Gly-Glu-Pro-Gln-pTyr-Gln-Pro-Lys(DNP)-Arg-NH 2 .…”