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A number of peptide substrates of the general structure Ac-Lxn-. . . -Lxz-Lxl-Gly-NH, have been synthesized and their a-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, E23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, K J~A (= 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 k , , l K g~ values indicates that the influence of a single structural change on the overall reactivity i s virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k,, and K E A 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: K E A or K E A and k,, change when glycine is introduced at the 22 position, when this glycine is replaced by alanine or when alanine is introduced at the 23 position.The observation that a-chymotrypsin has a marked side-chain specificity [l], in that it preferentially catalyzes the hydrolysis of peptide bonds C-terminal to aromatic amino-acid residues, led to the conclusion that the interactions between a single specific amino-acid residue and the active site of the enzyme are of primary importance in determining which peptide bond is to be cleaved. These interactions, which occur within what can be termed the primary interaction range, have therefore been investigated kinetically in detail using derivatives of single amino acids, mainly N-acylated esters, as model substrates I n the case of protein [8] and peptide [9] substrates, it appears that additional residues on both sides of the specific amino acid also interact with the active site. The importance of such additional interactions, which occur in the secondary interaction range, may be investigated by extending the kinetic studies to suitable peptide substrates. I n order to obtain kinetic data with peptides which is as accurate as that for ester substrates, it is necessary that only one peptide bond is hydrolyzed. For this purpose substrates of the general structure have been synthesized, where only Lxl is a specific amino acid residue. Thus it is expected that only the peptide bond between LZ1 and L,1 (indicated by the arrow) is cleaved during the measurements. In designing these substrates, it is important to select end groups Ex and E, which do not give rise to disturbing interactions with the enzyme.The pH-stat titration offers a simple method of analysis for ester hydrolysis. However, this method is technically more difficult to apply to peptide hydrolysis [lo], since the formation of amine product Em.

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...

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...