Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where HSA] ? 5×NphOAc] and NphOAc] ? 5×HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k_app and k_obs on HSA] and NphOAc], respectively, values of K_s, k₊₂, and k₊₂/K_s were determined. Values of K_s, k₊₂, and k₊₂/K_s obtained at HSA] ? 5×NphOAc] and NphOAc] ? 5×HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k₊₂/K_s, k₊₂, and K_s reflects the acidic pK_a shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.