Role of Glu318 and Thr319 in the catalytic function of cytochrome P450 d (P4501A2): effects of mutations on the methanol hydroxylation

Polar amino acids in the (putative) distal site are well conserved in P450s. For example, Glu318 for P450 d is well conserved as either Glu or Asp for P450s, and Thr319 for P450 d is also conserved for P450s. We have studied how mutations at Glu318 and Thr319 of P450 d influence the catalytic activity toward methanol associated with the activation of O 2 . Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type. O 2 consumption and NADPH oxidation rates of each mutants varied corresponding to the catalytic activities. However, surprisingly, efficiency (16–40%) of incorporated O to the substrate vs. consumed O 2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type. In addition, H 2 O 2 , which is produced from uncoupling for the wild‐type P450 d , was not observed for reaction of the Glu318Ala and Thr319Ala mutants. It seemed that consumed O 2 was partially reduced to 2 mol of H 2 O by 4‐electron transfer from NADPH for the wild‐type and Thr319Ala mutant. However, for the two Glu318 mutants, it appeared that the consumed O 2 was not reduced in the same way. It was thus suggested that the conserved Glu318 and Thr319 of P450 d are not essential for the activation of O 2 in the methanol oxidation. Role of the water molecule or the methanol molecule in the catalytic function was implied.—Hiroya, K.; Ishigooka, M.; Shimizu, T.; Hatano, M. Role of Gly318 and Thr319 in the catalytic function of cytochrome P450 d : effects of mutations on the methanol hydroxylation. FASEB J. 6: 749‐751; 1992.