Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps |
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Authors: | Paolo Ascenzi Magda Gioia Gabriella Fanali Massimo Coletta Mauro Fasano |
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Institution: | Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma, Italy; National Institute of Biostructures and Biosystems, Viale Medaglie d'Oro 305, I-00136 Roma, Italy. |
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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 kapp and kobs on HSA] and NphOAc], respectively, values of Ks, k+2, and k+2/Ks were determined. Values of Ks, k+2, and k+2/Ks 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/Ks, k+2, and Ks reflects the acidic pKa 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. |
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