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Journal of Biomolecular Screening
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*CUMENE HYDROPEROXIDE
*HYDROGEN PEROXIDE
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Article

Identification of a Novel Laser Dye Substrate of Mammalian Cytochromes P450: Application in Rapid Kinetic Analysis, Inhibitor Screening, and Directed Evolution

Santosh Kumar*

University of Texas Medical Branch

* To whom correspondence should be addressed. E-mail: sakumar{at}utmb.edu.


  Abstract
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) kcat =3 to 6 min-1, 2) Km, CuOOH =100 to 200 µM, and 3) S50, C152 =10 to 20 µM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high kcat (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. (Journal of Biomolecular Screening XXXX:xx-xx)

First published on May 3, 2007, doi:10.1177/1087057107301496

Journal of Biomolecular Screening 2007;12:677.

A more recent version of this article appeared on August 1, 2007

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