Document Detail

Roles of key active-site residues in flavocytochrome P450 BM3.
MedLine Citation:
PMID:  10191269     Owner:  NLM     Status:  MEDLINE    
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 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG 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 microM, kcat 14620 min-1; compared with values of 4.7 microM 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.
M A Noble; C S Miles; S K Chapman; D A Lysek; A C MacKay; G A Reid; R P Hanzlik; A W Munro
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  339 ( Pt 2)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1999 Apr 
Date Detail:
Created Date:  1999-06-23     Completed Date:  1999-06-23     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  371-9     Citation Subset:  IM    
Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, UK.
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MeSH Terms
Bacterial Proteins*
Base Sequence
Binding Sites
Cytochrome P-450 Enzyme System / chemistry,  genetics,  metabolism*
Cytochrome c Group / metabolism
DNA Primers
Electron Transport
Fatty Acids / metabolism
Flavins / metabolism
Heme / metabolism
Mixed Function Oxygenases / chemistry,  genetics,  metabolism*
NADPH-Ferrihemoprotein Reductase
Oxidoreductases / metabolism
Reg. No./Substance:
0/Bacterial Proteins; 0/Cytochrome c Group; 0/DNA Primers; 0/Fatty Acids; 0/Flavins; 14875-96-8/Heme; 9035-51-2/Cytochrome P-450 Enzyme System; EC 1.-/Mixed Function Oxygenases; EC 1.-/Oxidoreductases; EC Reductase; EC P450 BM3 monoxygenases

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