pH Dependency of the Zinc and Cobalt Carboxypeptidase Catalyzed Enolization of (R)-2-Benzyl-3-(p-methoxybenzoyl)propionic Acid

Article abstract of DOI:10.1021/ja00349a055

The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyl)propionic acid ((-)-1) has been determined.The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered.The pK_a values of 6.03 +/- 0.35 and 6.04 +/- 0.31 measured from the pH dependency of k_cat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pK_a for the ionization of the γ-carboxyl of Glu-270.The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate.The K_I-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occurs with pK_a = 7.56 +/- 0.15 for the Zn(II) enzyme and pK_a = 8.29 +/- 0.32 for Co(II) CPA while the other occurs above pH 9.The pK_a values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK.Finally, a reasonable interpretation of the pH dependency on a group with pK_a < 9 is that it corresponds to the ionization of the active-site metal ion bound water.