Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4

作     者：Szklarz, GD He, YQ Kedzie, KM Halpert, JR Burnett, VL 

作者机构：UNIV ARIZONA COLL PHARM DEPT PHARMACOL & TOXICOL TUCSON AZ 85721 USA 

出 版 物：《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 (生物化学与生物物理学集刊)

年 卷 期：1996年第327卷第2期

页      面：308-318页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NIEHS NIH HHS [ES03619  T-32 ES07091  P30 ES06694] Funding Source: Medline 

主　　题：cytochrome P450 mutagenesis steroid hydroxylation structure-function relationships heterologous expression molecular modeling 

摘      要：The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation, Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114 -- Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity. (C) 1996 Academic Press, Inc.