Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

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Abstract
The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K.F., Galdes, A., Martinelli, R.A., Holmquist, B., Auld, D.S. and Vallee, B.L. (1983) Biochemisty 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping." It allows peptide intermediates to be observed for longer periods (.mchgt. 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped with the cobalt enzyme (> 10 .mu.M) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is presented in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K''eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping to the ES2 intermediate allows analysis of its physiocochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra. Theoretical considerations and the present results suggest that similar trapping strategies should be applicable to intermediates of other hydrolytic enzymes.