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] >= 5x [NphOAc] and [NphOAc] 5x [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), obtained at [HSA] 5 x [NphOAc] and [NphOAc],>= 5 x [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, 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
Ascenzi, P., Gioia, M., Fanali, G., Coletta, M., Fasano, M. (2012). Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 424(3), 451-455 [10.1016/j.bbrc.2012.06.131].
Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps
ASCENZI, Paolo;
2012-01-01
Abstract
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] >= 5x [NphOAc] and [NphOAc] 5x [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), obtained at [HSA] 5 x [NphOAc] and [NphOAc],>= 5 x [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, 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 Tyr411I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
