dc.contributor.author | Noble, Michael A. | |
dc.contributor.author | Miles, Caroline S. | |
dc.contributor.author | Chapman, Stephen K. | |
dc.contributor.author | Lysek, Dominikus A. | |
dc.contributor.author | MacKay, Angela C. | |
dc.contributor.author | Reid, Graeme A. | |
dc.contributor.author | Hanzlik, Robert P. | |
dc.contributor.author | Munro, Andrew W. | |
dc.date.accessioned | 2011-10-31T15:58:45Z | |
dc.date.available | 2011-10-31T15:58:45Z | |
dc.date.issued | 1999 | |
dc.identifier.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). | |
dc.identifier.uri | http://hdl.handle.net/1808/8315 | |
dc.description | Abbreviations used: P450, cytochrome P450 mono-oxygenase; ImC12, 12-(imidazolyl)dodecanoic acid; 1-PIM, 1-phenylimidazole. | |
dc.description.abstract | 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. | |
dc.language.iso | en_US | |
dc.publisher | Portland Press | |
dc.relation.isversionof | http://www.biochemj.org/bj/339/bj3390371.htm | |
dc.subject | Inhibition | |
dc.subject | Kinetics | |
dc.subject | Mutagenesis | |
dc.title | Roles of key active-site residues in flavocytochrome P450 BM3 | |
dc.type | Article | |
kusw.kuauthor | Hanzlik, Robert P. | |
kusw.kudepartment | Department of Medicinal Chemistry | |
kusw.oastatus | fullparticipation | |
dc.identifier.doi | 10.1042/bj3390371 | |
kusw.oaversion | Scholarly/refereed, publisher version | |
kusw.oapolicy | This item meets KU Open Access policy criteria. | |
dc.rights.accessrights | openAccess | |