Sequence requirements for cytochrome P-450IID1 catalytic activity. A single amino acid change (Ile380 Phe) specifically decreases Vmax of the enzyme for bufuralol but not debrisoquine hydroxylation.
A cDNA coding for an allelic variant of rat IID1, designated IID1v, was isolated that produced a P-450 having a 10-fold lower catalytic activity toward the substrate bufuralol when expressed in COS-1 cells (Matsunaga, E., Zanger, U. M., Hardwick, J. P., Gelboin, H. V., Meyer, U. A., and Gonzalez, F. J. (1989) Biochemistry, 28, 7349-7355). IID1 and IID1v cDNA-deduced proteins differed in sequence by 4 amino acid residues. IID1 has Val, Phe, Arg, and Leu while IID1v has Ile, Leu, Gln, and Phe at amino acid positions 123, 124, 173, and 380, respectively. Chimeric cDNAs between IID1 and IID1v were constructed and expressed in hepatoma cells using vaccinia virus. A chimera having the Phe (IID1v) at amino acid 380, with the remaining 3 variant amino acid residues of IID1, was found to have a 17-fold decrease in Vmax and a 2 to 3-fold decrease in Km for (+)-bufuralol 1'-hydroxylation when compared to a converse chimera having Ile (IID1) in a background of IID1v sequence. Although this enzyme lacked significant bufuralol metabolism, it was able to carry out debrisoquine 4-hydroxylation. In contrast, the chimera having Ile (IID1) at position 380 was lacking in debrisoquine 4-hydroxylation. Type I difference spectra analysis revealed that both forms could bind debrisoquine with similar spectral dissociation constants. These data demonstrate that the single amino acid substitution Ile380----Phe differentially decreases the catalytic activity of IID1 toward bufuralol but not debrisoquine.
Matsunaga
E
E
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Zeugin
T
T
Zanger
U M
UM
Aoyama
T
T
Meyer
U A
UA
Gonzalez
F J
FJ
D000319:Adrenergic beta-Antagonists / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D003647:Debrisoquin / Q000378:metabolism*; D004983:Ethanolamines / Q000378:metabolism*; D006900:Hydroxylation; D007532:Isoleucine; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D051381:Rats; D011995:Recombination, Genetic; D013053:Spectrophotometry; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000235:genetics
D000319:Adrenergic beta-Antagonists; D004273:DNA, Neoplasm; D004983:Ethanolamines; D007532:Isoleucine; D009249:NADP; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D003647:Debrisoquin