Effect of mutations at Lys250, Arg251, and Lys253 of cytochrome P450 1A2 on the catalytic activities and the bindings of bifunctional axial ligands.
Some eukaryotic cytochromes P450 (P450s) have a series of ionic amino acids, corresponding to Lys250, Arg251, and Lys253 residues in the P450 1A2 sequence. To understand the roles of those ionic amino acids in the catalytic function of P450, three single mutants, Lys250Leu, Arg251Leu, and Lys253Leu of P450 1A2 were obtained from yeast (Saccharomyces cerevisiae) expression system. Turnover numbers of the Arg251Leu mutant in dealkylation reactions of methoxy- and ethoxyresorufin catalyzed by the P450 reconstituted system were remarkably increased by sixfold compared to those of the wild type. The Lys250Leu and Lys253Leu mutants also showed turnover numbers higher than those of the wild type by three- to fourfold. Those catalytic activities were inhibited competitively by pyridine derivatives, nitrogenous axial ligands to the P450 heme. From those findings together with other spectral data, it was suggested that the ionic site of Lys250, Arg251, and Lys253 may be somehow located near the substrate recognition site and/or near the axial-ligand access channel of this enzyme.
Krainev
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Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Shimizu
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Hiroya
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Hatano
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D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D008024:Ligands; D010078:Oxazines; C076580:7-methoxyresorufin; C007649:ethoxyresorufin; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D008239:Lysine