ROLE OF THR-252 IN CYTOCHROME P450(CAM) - A STUDY WITH UNNATURAL AMINO-ACID MUTAGENESIS

Replacement of Thr-252 in the active center of cytochrome P450cam with a non-hydroxy amino acid residue such as Ala and Val by conventional site-directed mutagenesis converted this monooxygenase to an NADH oxid ase (Imai, M. et al. Proc. Natl. Sci. U. S. A. 86, 7823-7827, 1989). I n this study, a mutant enzyme with a methoxy group in place of the hyd roxy group of Thr-252 (OMe-mutant) was synthesized by the method of un natural amino acid mutagenesis (Noren, C. J. et al., Science 244, 182- 188, 1989). Unlike other site-directed mutants without a hydroxy group at the position, the OMe-mutant retained a considerably high monooxyg enase activity, yielding a stoichiometric amount of 5-exo-hydroxycamph or to that of the oxygen consumed. Thus a free hydroxy group at this p osition is not an indispensable requisite for the monooxygenase to cle ave the O-O bond of molecular O-2 as previously proposed. (C) 1995 Aca demic Press. Inc.