title: Topological alteration of the CYP3A4 active site by the divalent cation Mg(2+).
abstract: Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of four N-phenyl-protoporphyrin IX isomers (N(B):N(A):N(C):N(D), 01:01:02:02). In the presence of 10 mM MgCl(2), the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N(A) isomer (N(B):N(A):N(C):N(D), 01:34:01:02). In addition, an investigation of MgCl(2) effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl(2) increased the apparent K(m) of triazolam 4-hydroxylation from 83 to 173 microM and reduced the V(max) for the reaction from 3.4 to 2.4 min(-1). Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl(2), it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 +/- 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl(2), the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 +/- 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity.
authors.affiliation: Drug Metabolism Research, Pharmacia Corporation, Kalamazoo, Michigan, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Schrag
authors.forename: L C
authors.initials: LC
authors.lastname: Wienkers
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007097:Imines / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011524:Protoporphyrins / Q000378:metabolism; D011721:Pyrenes / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014229:Triazolam / Q000378:metabolism
chemical_list: D007097:Imines; D011524:Protoporphyrins; D011721:Pyrenes; D014229:Triazolam; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; C030984:pyrene; C028025:protoporphyrin IX; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008274:Magnesium