Substrate Routes to the Buried Active Site May Vary among Cytochromes P450: Mutagenesis of the F-G Region in P450 2B1
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- 作者：Emily E. Scott ; You Qun He ; and James R. Halpert
- 刊名：Chemical Research in Toxicology
- 出版年：2002
- 出版时间：November 2002
- 年：2002
- 卷：15
- 期：11
- 页码：1407 - 1413
- 全文大小：114K
- 年卷期：v.15,no.11(November 2002)
- ISSN：1520-5010
文摘
Until recently, all known structures of bacterial cytochromes P450 suggested that substrateaccess to the buried active site occurred via the F-G region, a surface loop distal to the hemecavity. However, the structure of P450 51 indicates a large opening from the protein surfacealong the I helix N-terminus, at right angles to the F-G channel. The single availablemicrosomal P450 structure (2C5) does not obviously favor one potential access route over theother. To determine whether the F-G region forms part of the substrate access channel in themicrosomal cytochrome P450 2B1, 11 residues between positions 208 and 230 were substitutedwith smaller and larger side chains in a highly expressed truncated form of the enzyme. Steady-state kinetic parameters were determined with the substrates testosterone, 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), and 7-benzyoxyresorufin (7-BR). The largest changes, 2-6-fold increases in kcat with testosterone and 7-EFC, were observed for L209A, which also exhibitsan altered testosterone metabolite profile and probably forms part of the active site roof. F219Wdemonstrated little or no activity with any of the three substrates examined, although the Ksvalue for benzphetamine binding was unaltered. S221F showed little activity with 7-BR. Nosignificant changes were observed in Km(testosterone) or S50(7-EFC) values for any of themutants, in stark contrast to the 10-fold and 100-fold changes in Km observed for mutants inthis region of other cytochromes P450. The minimal changes in 2B1 do not support access viathe F-G region of 2B1 and suggest the alternate access route identified in P450 51.