Transition state structure determination : analysis of the hydration of 2-acetyl-3, 4-dimethylthiazolium ion and [alpha]-chymotrypsin catalyzed hydrolyses using the proton inventory technique

Abstract

The equilibrium hydration of 2-acetyl-3,4-dimethylthiazolium ion was investigated in water-acetonitrile mixtures by using the proton inventory technique and by measuring thermodynamic activation parameters. The solvent isotope effect, k(,H(,2)O)/k(,D(,2)O), on the rate constant, k(,f), for the forward step of the equilibrium hydration varied from 3.10 to 2.58 as the volume fraction of acetonitrile was increased from 0.25 to 0.90. The solvent isotope effects were found to be consistent with a transition state consisting of two water molecules in which one water molecule acts as a general base to abstract a proton from the nucleophilic water molecule. The transition state was found to be invariant within this range of solvent composition. The kinetics of the deacylation step of the (alpha)-chymotrypsin and 1-methyl-His-57 chymotrypsin catalyzed hydrolysis of p-nitrophenyl acetate, p-nitrophenylthiolacetate, N-Cbz-glycine-p-nitrophenyl ester, and N-Cbz-glycyl-glycine-p-nitrophenyl ester were investigated by using the proton inventory technique. The proton inventories for the native (alpha)-chymotrypsin catalyzed hydrolysis of the minimal substrates p-nitrophenyl acetate and p-nitrophenylthiolacetate were consistent with one proton giving rise to the observed solvent isotope effects of 2.30 and 1.91, respectively. With the methylated enzyme, these isotope effects changed to 1.05 and 1.50, and the proton inventories were consistent with proton transfer no longer being important in the rate-limiting process. The substrates N-Cbz-glycine-p-nitrophenyl ester and N-Cbz-glycyl-glycine-p-nitrophenyl ester gave proton inventories for catalysis by the native enzyme which were consistent with two protons in the rate-limiting process of deacylation giving rise to the solvent isotope effects of 2.76 and 2.42. The hydrolysis of N-Cbz-glycine-p-nitrophenyl ester by the methylated enzyme showed the transfer of a single proton, with an isotope effect of 2.38, coupled with a partially rate-limiting, nonisotopic step being important in catalysis. These results were interpreted in terms of a mechanism involving initial acylation of the imidazole ring of His-57 with the substrates p-nitrophenyl acetate and p-nitrophenylthiolacetate, and by a mechanism involving the unmethylated nitrogen of His-57 acting as a general base in catalysis of the amino acid substrates by methylchymotrypsin.