Chimeric cDNA expression and site directed mutagenesis studies of cytochrome P450s CYP2A1 and CYP2A2.
Construction of chimeras and site directed mutagenesis were used to study the regioselectivity and kinetics of testosterone hydroxylation by the cytochrome P450s CYP2A1 and CYP2A2. Although these enzymes exhibit 88% sequence similarity, they catalyze very different regioselective hydroxylations of testosterone. Active chimeras inwhich the first 355 amino acids do not correspond to a single enzyme show broad radioselectivity, whereas the specificity of the parent enzyme is obtained if the first 355 amino acids are unchanged. Therefore, the region between amino acids 275 and 355 is important in maintaining regioselectivity. Single point mutants were constructed for the 13 amino acid differences in this region. For 26 single point and 2 double mutants all active mutants have the same regioselectivity as the parent enzymes. However, kinetic analysis of the CYP2A1 mutants showed that 4 single point mutants and 1 double mutant had kinetic parameters very different from the parent enzyme. All of these substitutions are associated with the conserved dioxygen binding region of the putative I helix predicted from the crystal structure of P450(cam). Deuterium isotope effects were used to determine any changes in the rate of reduction and to estimate the relative amount of excess water formation. Changes in reduction rates are not sufficient to account for the differences in V(max) values. Therefore, it is likely that the amount of hydrogen peroxide formed is a primary determinant of V(max).
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
N
N
Hanioka
F J
FJ
Gonzalez
N A
NA
Lindberg
G
G
Liu
K R
KR
Korzekwa
D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000378:metabolism; D003903:Deuterium; D005260:Female; D057927:Hydrophobic and Hydrophilic Interactions; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D018076:DNA, Complementary; D050506:Mutant Chimeric Proteins; D050505:Mutant Proteins; D013739:Testosterone; D003903:Deuterium; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C456179:Cyp2a1 protein, rat; D000072467:Cytochrome P450 Family 2; C050356:steroid 15-alpha-hydroxylase