Roles of key active-site residues in flavocytochrome P450 BM3
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Issue Date
1999Author
Noble, Michael A.
Miles, Caroline S.
Chapman, Stephen K.
Lysek, Dominikus A.
MacKay, Angela C.
Reid, Graeme A.
Hanzlik, Robert P.
Munro, Andrew W.
Publisher
Portland Press
Type
Article
Article Version
Scholarly/refereed, publisher version
Published Version
http://www.biochemj.org/bj/339/bj3390371.htmMetadata
Show full item recordAbstract
The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DG‡) resulting from a smaller DG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.
Description
Abbreviations used: P450, cytochrome P450 mono-oxygenase; ImC12, 12-(imidazolyl)dodecanoic acid; 1-PIM, 1-phenylimidazole.
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Citation
M. A. Noble, C. S. Miles, S. K Chapman, D. A. Lysek, A. Madkay, G. A. Reid, R. P. Hanzlik and A. W. Munro, "The roles of key active site residues in flavocytochrome P450." Biochemical Journal, 339, 371-379 (1999).
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