Received for publication June 22, 2005.
Revised August 25, 2005.
Accepted for publication August 26, 2005.

Hydrolysis of Angiotensin II Receptor Blocker Prodrug Olmesartan Medoxomil by Human Serum Albumin and Identification of Its Catalytic Active Sites

Abstract

In the present study, we investigated the esterase-like activity of human serum albumin (HSA) and the mechanism by which it hydrolyzes, and thereby activates, olmesartan medoxomil (CS-866), a novel angiotensin II receptor antagonist. CS-866 has previously been shown to be rapidly hydrolyzed in serum in which HSA appeared to play the most important role in catalyzing the hydrolysis. We found that the hydrolysis of CS-866 by HSA followed Michaelis-Menten kinetics. Compared with the release of p-nitrophenol from p-nitrophenyl acetate (PNPA), CS-866 showed smaller affinity to HSA and slower catalytic rate of hydrolysis. Thermodynamic data indicated that PNPA has a smaller value of activation entropy (S) than CS-866; consequently, PNPA is more reactive than CS-866. Ibuprofen and warfarin acted as competitive inhibitors of hydrolysis of CS-866, whereas Dansyl-L-asparagine (DNSA), n-butyl p-aminobenzoate (n-butyl p-AB) and diazepam did not. These findings suggest that the hydrolytic activity is associated to parts of site I and site II for ligand binding. All chemically modified HSA derivatives (Tyr-, Lys-, His- and Trp-modifications) had significantly lower reactivity than native HSA; Lys-HSA and Trp-HSA had especially low reactivity. All the mutant HSAs tested (K199A, W214A and Y411A) exhibited a significant decrease in reactivity, suggesting that Lys-199, Trp-214 and Tyr-411 play important roles in the hydrolysis. Results obtained using a computer docking model are in agreement with the experimental results, and strongly support the hypotheses that we derived from the experiments.

Key words: enzyme kinetics, ligand docking, plasma protein binding, protein binding, recombinant proteins, site-directed mutagenesis

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