Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics.
The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.
Koley
A P
AP
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Robinson
R C
RC
Friedman
F K
FK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A