Relaxation spectra of proteinases. Isomerizations of carboxypeptidase A(Cox) and (Anson)

Abstract

Carboxypeptidase A (Cox) isomerizes at 25° and at neutral pH values in the absence of substrates. The relaxation effect of largest amplitude (τ₁), detected with proton indicators by the equilibrium temperature-jump method, is characterized by pH-dependent rate constants varying from 12 msec^-1 in acid to 6 msec^-1 in alkaline solution, and by a single pK_a value near 6. The τ₁ effect is also present in the commercial Worthington enzyme and in the isomers A_γ^Leu, A_γ^Val, and A_β^Leu The pH-dependent behavior of the isomerization can be interpreted in terms of the three-protonation-state model for the enzyme (Auld, D. S., and Vallee, B. L. (1970), Biochemistry 9, 4352) and therefore may be linked to the ionization of a group important to the catalytic step of peptide hydrolysis. The transient-state behavior of noncompetitive inhibitors of peptide hydrolysis is correlated with the analogous steady-state behavior. The τ₁ effect is not observed at inhibitor concentrations sufficient to saturate the enzyme under steady-state conditions. Rate constants greater than 12 msec^-1 are observed in the presence of saturating concentrations of glycyl-l-tyrosine, a pseudosubstrate, and l-phenylalanine, a product and competitive inhibitor of peptide hydrolysis, whereas no change in the relaxation time or amplitude occurs upon addition of l-phenyllactate and l-mandelate, products and competitive inhibitors of ester hydrolysis. The relevance of these results to a proposal of nonidentical binding sites for peptide and ester substrates of carboxypeptidase (Vallee et al. (1968), Biochemistry 7, 3547) is discussed.