Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity.
Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-450 and NADPH:cytochrome P-450 reductase domains, that catalyses the hydroxylation of fatty acids. The natural ferriprotoporphyrin IX has been removed from the haem domain of cytochrome P-450 BM-3 by treatment with acidified acetone, and it has been shown that, under carefully controlled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, substrate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporphyrin IX dimethyl ester yields a protein which has a higher affinity for the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacement with ferrimesoporphyrin IX yields a protein with the same affinity for substrate, but a reduced catalytic turnover. These results suggest that the haem moiety has a role in the creation of the binding pocket for substrate, and that modification of the electron density on the haem iron effects the catalytic rate.
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Department of Biochemistry, University of Leicester, U.K.
Primrose
W U
WU
Lian
L Y
LY
Roberts
G C
GC
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006418:Heme / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013053:Spectrophotometry
D001426:Bacterial Proteins; D006418:Heme; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases