Esterase-like Activity of Human Serum Albumin. VI. Reaction with p-Nitrophenyl Glycinate

Abstract

The reaction of p-nitrophenyl glycinate (NPG, pK_a=7.1) with human serum albumin (HSA) was investigated kinetically at various pH's and 25°C. The Michaelis constant (K_s/m in Nt, where m is the number of reactive sites) and the catalytic rate constant (k₂ in s^-1) were determined. The affinity of the cationic substrate (NPGH⁺) to the reactive site is lower than that of the neutral substrate (NPG). The pH profile of k₂ is sigmoidal, like that of k_o, indicating ionization of the reaction species at pH 6-7. The effect of chemical modification of histidine residues by diethylpyrocarbonate on the reaction rate with NPG and the deuterium oxide isotope effect on the reaction rate of HSA with NPG indicate the reaction of NPG HSA complex with OH^- rather than the nucleophilic reaction (formation of glycinated HSA) of the histidine imidazole group (s) with NPG. Results on the reaction in the presence of excess NPG over HSA suggest that HSA has nonspecific multiple reactive sites towards NPG. The reactive sites were considered to be relatively exposed on the surface of the HSA molecule and to be sensitive to conformational change.