Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 455, Issue 1, Pages 61-67Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2006.08.024
Keywords
cytochrome P450; structure function relationships; site-directed mutagenesis; enzyme catalysis and inhibition; P450 2134 crystal structure
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Funding
- NIEHS NIH HHS [ES 03619, R01 ES003619, P30 ES006676, ES 06676] Funding Source: Medline
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Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2134, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a > 4-fold decrease in differential inhibition by BIT and 4-CPI (IC50,BIF/IC50,4-CPI). F296A, T302A, I363A, V367A, and V477A showed a >= 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K-m. T302A, V367L, and V477A showed > 4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered sterco- and regioselectivity. Interestingly, I363A showed a >= 150-fold enhanced k(cat)/K-m with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis. Published by Elsevier Inc.
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