Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.
Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.
Hanioka
N
N
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Gonzalez
F J
FJ
Lindberg
N A
NA
Liu
G
G
Gelboin
H V
HV
Korzekwa
K R
KR
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics
D011994:Recombinant Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C029009:steroid hormone 7-alpha-hydroxylase