title: Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps.
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]≥ 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.
authors: Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma, Italy. ascenzi@uniroma3.it
authors: Paolo
authors: P
authors: Ascenzi
authors: Magda
authors: M
authors: Gioia
authors: Gabriella
authors: G
authors: Fanali
authors: Massimo
authors: M
authors: Coletta
authors: Mauro
authors: M
authors: Fasano
mesh_terms: D002384:Catalysis; D004950:Esterases / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D009596:Nitrophenols; D012709:Serum Albumin; D014443:Tyrosine; C008642:4-nitrophenyl acetate; D004950:Esterases