STRUCTURAL DETERMINANTS OF CYTOCHROME-P450-2B1 SPECIFICITY - EVIDENCEFOR 5 SUBSTRATE RECOGNITION SITES

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed ov er seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined us ing androstenedione and testosterone as substrates. Substitutions at p ositions 303, 360, and 473 did not markedly affect the regio- or stere oselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly diffe rent steroid metabolite profiles compared with parental P450 2B1. In p articular, the Phe-206 --> Leu substitution conferred androgen 6alpha- and testosterone 7alpha-hydroxylase activities, and the Thr-302 --> S er substitution suppressed androgen 16beta-hydroxylation in favor of a ndrostenedione 16alpha- and testosterone 15alpha-hydroxylation. Replac ement of Val-363 or Val-367 with Ala conferred androgen 15alpha-hydrox ylase and 6beta-hydroxylase activities, respectively, and suppressed s usceptibility to mechanism-based inactivation by the P450 2B1-selectiv e chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367 --> Ala mutant was also resistant to chloramphenicol itsel f. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most in terestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face a nd hydroxylation on the D-ring or the B-ring. The findings indicate th e importance of residues 206, 302, 363, and 367 in P450 2B1 in determi ning substrate specificity and regio- or stereoselectivity and, togeth er with previous studies of residues 114 (SRS-1) and 478 (SRS-6), prov ide experimental evidence for the existence of at least five substrate recognition sites in P450 2B1.