Identification of a novel laser dye substrate of mammalian cytochromes P450: application in rapid kinetic analysis, inhibitor screening, and directed evolution.
The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)-supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) k(cat) = 3 to 6 min(-1), 2) K(m, CuOOH) = 100 to 200 microM, and 3) S(50, C152) = 10 to 20 microM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high k(ca)t (27 and 38 min(-1), respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)-supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution.
Kumar
Santosh
S
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555, USA. sakumar@utmb.edu
D000818:Animals; D001555:Benzene Derivatives / Q000378:metabolism / Q000494:pharmacology; D002021:Buffers; D004396:Coloring Agents / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D008099:Liver / Q000201:enzymology; D008322:Mammals; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001555:Benzene Derivatives; D002021:Buffers; D004396:Coloring Agents; D003374:Coumarins; D016877:Oxidants; D011994:Recombinant Proteins; C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; C007164:cumene hydroperoxide