Decreased substrate affinity upon alteration of the substrate-docking region in cytochrome P450(BM-3).
A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis. The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates. The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme. In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form.
Maves
S A
SA
Beckman Institute for Advanced Science and Technology and Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Yeom
H
H
McLean
M A
MA
Sligar
S G
SG
D001120:Arginine; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D005973:Glutamine; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D019308:Palmitic Acid / Q000378:metabolism; D017354:Point Mutation; D011392:Proline; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D007850:Lauric Acids; D009227:Myristic Acids; D011994:Recombinant Proteins; D019814:Myristic Acid; D005973:Glutamine; C030358:lauric acid; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D011392:Proline; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases