ROLE OF RESIDUE-478 AS A DETERMINANT OF THE SUBSTRATE-SPECIFICITY OF CYTOCHROME-P450-2B1

被引:71
作者
HE, YA [1 ]
BALFOUR, CA [1 ]
KEDZIE, KM [1 ]
HALPERT, JR [1 ]
机构
[1] UNIV ARIZONA, COLL PHARM, DEPT PHARMACOL & TOXICOL, TUCSON, AZ 85721 USA
关键词
D O I
10.1021/bi00153a015
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16beta-OH:16alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16beta-OH:16alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16beta-hydroxylase activity, and the 16beta-OH:16alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation. Overall, this study demonstrates the importance of the amino acid at position 478 in dictating the substrate specificity of P450 2B1 and provides firm experimental evidence for a model in which this residue comprises part of a substrate recognition site.
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页码:9220 / 9226
页数:7
相关论文
共 34 条
[1]  
AOYAMA T, 1989, J BIOL CHEM, V264, P21327
[2]  
CAJACOB CA, 1988, J BIOL CHEM, V263, P18640
[4]  
CULLEN BR, 1987, METHOD ENZYMOL, V152, P684
[5]   PRIMARY STRUCTURE OF A CYTOCHROME-P-450 - CODING NUCLEOTIDE-SEQUENCE OF PHENOBARBITAL-INDUCIBLE CYTOCHROME-P-450 CDNA FROM RAT-LIVER [J].
FUJIIKURIYAMA, Y ;
MIZUKAMI, Y ;
KAWAJIRI, K ;
SOGAWA, K ;
MURAMATSU, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1982, 79 (09) :2793-2797
[6]  
GOTOH O, 1992, J BIOL CHEM, V267, P83
[7]   EVIDENCE FOR FUNCTIONAL AND STRUCTURAL MULTIPLICITY OF PREGNENOLONE-16-ALPHA-CARBONITRILE-INDUCIBLE CYTOCHROME-P-450 ISOZYMES IN RAT-LIVER MICROSOMES [J].
GRAVES, PE ;
KAMINSKY, LS ;
HALPERT, J .
BIOCHEMISTRY, 1987, 26 (13) :3887-3894
[8]  
HALPERT J, 1990, DRUG METAB DISPOS, V18, P168
[9]  
HALPERT J, 1983, MOL PHARMACOL, V23, P445