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The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

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In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The K , and k,, for the different substrates were determined by standard steady-state kinetics, and the corresponding AAGTx value derived from k,,/K,,, was used for the comparison, setting AAGTt for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions l', 2: or 3' (AAG,t values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and 0-Me ester derivatives caused a change in AAGTx values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position. Thus, while the peptides with single Asp or Phe substitutions in l', 2: and 3' gave AAGTr values of 3-5 kJ, the peptide containing all 3 modifications, Ac-ADDF, gave only 1 kJ. Similarly, Ac-TAAA was a poor substrate and Ac-GAAA was not cleaved at all in this study, while in the past other peptides such as Ac-TGG and Ac-GGG have been found to be excellent and reasonably good substrates, respectively. Although the rate differences observed in this work are minor, they nevertheless appear to reflect the kind of structural detail that is involved in determining the specificity of protein-protein (peptide) interactions.

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Renin, the rate-limiting enzyme in the formation of angiotensin II, is well-known for its stringent substrate specificity. In this study, the biochemical basis for the unusual specificity of renin was investigated by replacing individual amino acids in the octapeptide substrate of renin with Ala. Kinetic analyses of Ala-substituted substrates revealed that the substitutions did not cause significant changes in the Km values, but did cause variable changes in the kcat and kcat/Km values. Ala substitutions at the P1', P1, and P3 sites decreased the kcat/Km values by 400-700-fold. Similar substitutions at the P3', P2, P4, and P5 sites only reduced the kcat/Km values by 2-7-fold. Interestingly, Ala substitution for the P2' Val produced a substrate with an approximately 3-fold increase in activity. These results indicate that the P1', P1, and P3 residues are crucial in determining the substrate specificity of renin. The findings also suggest that the specificity of renin is achieved mainly through substrate discrimination in the transition state, rather than in the ground state. Further studies on the effects of amino acid substitutions at the P2' site revealed that non-branched-chain amino acids (e.g., Ala and alpha-aminobutyric acid) are preferred at this site. Only P1' substitution demonstrated any significant change in Km, presumably due to the decreased hydrophobic interactions in the S1' site upon Ala substitution. The species specificity of renin presumably arises from differing P1'-P3' residues in angiotensinogens. For example, the P1'-P3' residues from human and porcine angiotensinogens are Ile-Val-His and Leu-Val-Tyr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)