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

Abstract

Polar amino acids in the (putative) distal site are well conserved in P450s. For example, Glu318 for P450d is well conserved as either Glu or Asp for P450s, and Thr319 for P450d is also conserved for P450s. We have studied how mutations at Glu318 and Thr319 of P450d influence the catalytic activity toward methanol associated with the activation of O2. Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type. O2 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 O2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type. In addition, H2O2, which is produced from uncoupling for the wild-type P450d, was not observed for reaction of the Glu318Ala and Thr319Ala mutants. It seemed that consumed O2 was partially reduced to 2 mol of H2O by 4-electron transfer from NADPH for the wild-type and Thr319Ala mutant. However, for the two Glu318 mutants, it appeared that the consumed O2 was not reduced in the same way. It was thus suggested that the conserved Glu318 and Thr319 of P450d are not essential for the activation of O2 in the methanol oxidation. Role of the water molecule or the methanol molecule in the catalytic function was implied.

Selection of Human Cytochrome P450 1A2 Mutants with Enhanced Catalytic Activity for Heterocyclic Amine N-Hydroxylation

“…Interestingly, substitution with the acidic residue Glu did not alter the activity of SF513 either (Table 4). A similar result was observed in the site-directed mutagenesis study of rat P450 1A2 in which Glu and Asp changes at residue 318 yielded different catalytic activities (61). The present result may exclude the possibility that the Asp437 residue exerts a role through the ionic interaction in the structural moiety, although the distance for binding may be an issue.…”

Oxidations of p-Alkoxyacylanilides Catalyzed by Human Cytochrome P450 1A2: Structure−Activity Relationships and Simulation of Rate Constants of Individual Steps in Catalysis

“…The mutant is not impaired in its ability to bind (thiol-liganded) heme or its rate of reduction to the ferrous form (12). This mutant is analogous to the rat P450 1A2 E318A mutant (and other Glu318 mutants) studied extensively by Shimizu and his associates (28)(29)(30)(69)(70)(71) and to (bacterial) P450 101 Asp 251 mutants (68,72,73). 7 This mutant, in contrast to mutant E225I with enhanced activity, presented a number of problems in transposing the mechanism and kinetic constants used for wild-type P450 1A2.…”

Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

“…Incorporation of CPR into preformed and purified CYP3A4-Nanodiscs was made by direct addition of oligomeric CPR at 1:4 CYP3A4/CPR molar ratio, as described [19]. All experiments have been performed at 37 °C using POPC Nanodisc system similar to our earlier detailed mechanistic studies [11-13] in order to allow direct comparison of results. This reconstitution system provides a stable well characterized and monodisperse preparation of CYP3A4 incorporated into the model lipid bilayer effectively mimicking the native membrane.…”