Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.
An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177-185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.
Renaud
J P
JP
CNRS URA 400, Universite Rene Descartes, Paris, France.
Davydov
D R
DR
Heirwegh
K P
KP
Mansuy
D
D
Hui Bon Hoa
G H
GH
D000818:Animals; D002151:Calorimetry; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D019277:Entropy; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry; D013816:Thermodynamics
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A