Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W.
The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S(50)=5.4 microM, n=1.0) but retained cooperativity of alpha-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K(D)=0.3 microM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6beta-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.
Fernando
Harshica
H
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA.
Davydov
Dmitri R
DR
Chin
Christopher C
CC
Halpert
James R
JR
D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D001971:Bromocriptine / Q000737:chemistry / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006420:Hemeproteins / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D011374:Progesterone / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
C463480:1-pyrenebutanol; D001059:Apoproteins; D006420:Hemeproteins; D021122:Protein Subunits; D011721:Pyrenes; D001971:Bromocriptine; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human