Catalytic properties of an expressed cytochrome P450 2B1 from a Wistar-Kyoto rat liver cDNA library.
Cytochrome P450 2B1 clones were isolated from a phenobarbital-induced Wistar-Kyoto (WKY) hepatic cDNA library and were found to contain a Glu-322 --> Val substitution, compared with wild-type 2B1 from Sprague-Dawley rats. After heterologous expression in Escherichia coli and purification, activities of this 2B1 E322V variant were determined for ethoxycoumarin and androstenedione. The total activities and metabolite profiles did not differ between 2B1 E322V and wild-type 2B1 for these substrates. In addition, similar rate constants of inactivation were observed with the mechanism-based inactivators chloramphenicol, N-(2-p-nitrophenethyl)chlorofluoroacetamide, and 9-ethynylphenanthrene. These results suggest that the Glu-322 --> Val alteration in the 2B1 WKY variant does not significantly affect 2B1 activity. However, another clone obtained from the cDNA library contained two additional substitutions: Val-103 --> Ala and Glu-424 --> Lys. As residue 103 is within a predicted substrate recognition site (SRS-1), it was of interest to determine whether the Val --> Ala substitution conferred any unique catalytic activities on 2B1. No differences in the metabolism of ethoxycoumarin or androstenedione were observed. However, the Val-103 --> Ala alteration caused an approximately threefold decrease in the rate constant of inactivation for 9-ethynylphenanthrene in comparison with either 2B1 E322V or wild-type 2B1. Based on computer modeling, residue 103 is predicted to be near the active site but at a distance greater than 5A from 9-ethynylphenanthrene. Our results suggest that the Val-103 --> Ala alteration may have an indirect influence on the susceptibility of P450 2B1 to mechanism-based inactivators.
Kobayashi
Y
Y
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, USA.
Strobel
S M
SM
Hopkins
N E
NE
Alworth
W L
WL
Halpert
J R
JR
D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D019362:Cytochrome P-450 CYP2B1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D011921:Rats, Inbred WKY
D018076:DNA, Complementary; D019362:Cytochrome P-450 CYP2B1