Effect of mutations of ionic amino acids of cytochrome P450 1A2 on catalytic activities toward 7-ethoxycoumarin and methanol

Abstract

Catalytic efficiencies, percentages of rates of product formation per NADPH oxidized, and rates of product formation per O2 consumed of ionic mutants of cytochrome P450 1A2 (P450 1A2) were studied. Efficiencies of Lys99Glu, Lys453Glu, and Arg455Glu mutants for the hydroxylation reaction toward 7-ethoxycoumarin in the reconstituted system were much lower than that of the wild type (less than 17%), which corresponds to lower turnover numbers for these mutants. In contrast, the catalytic efficiencies for the hydroxylation reaction toward methanol of the three mutants were more than 45% that of the wild type in spite of these mutants' lower turnover numbers. Turnover numbers and catalytic efficiencies of Arg137Leu and Lys401 Glu mutants toward both substrates were comparable to those of the wild type. The electron-transfer rate from the reductase to the heme of P450 1A2 was decreased by 30% upon addition of excess methanol, while it was not influenced by addition of excess 7-ethoxycoumarin. The turnover numbers toward both 7-ethoxycoumarin and methanol as well as the rate constant of electron transfer were decreased by 25-40% by raising the concentration of KCl from 0 to 300 mM in the reconstituted system containing 50 mM potassium phosphate buffer. The turnover numbers toward both substrates of the above-mentioned five ionic mutants caused by tert-butyl hydroperoxide in the absence of the reductase and NADPH were comparable to those of the wild type. The effect of phospholipid constituents on the catalytic activity toward 7-ethoxycoumarin of the wild type was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme–substrate binding

“…CYP1A2 oxidation of EOMCC was 92.1% uncoupled for all buffer concentrations and temperatures at pH 6.7. Similarly high uncoupling percentages have been found for the CYP1A2 oxidation of phenacetin [29, 34] and of 7-ethoxycoumarin [35] and for other human P450 enzymes [36–38]. A much weaker correlation between -r EOMCC and -r NADPH was found as a function of buffer type and pH (Fig 5B) indicating that coupling is altered by these variables.…”

Crucial Role of Lys423 in the Electron Transfer of Neuronal Nitric-oxide Synthase

“…nNOS was expressed in Saccharomyces cerevisiae using the acid phosphatase promoter previously used for the expression of cytochrome P450 1A2 (25)(26)(27). The oligonucleotide primers for the mutations of Lys 423 to Glu, Met, Leu, and Asn were 5Ј-CCAGTGGTCCGAGCTGCA-GG-3Ј, 5Ј-CAGTGGTCCATGCTGCAGGT-3Ј, 5Ј-CAGTGGTCCCTGCT-GCAGGT-3Ј, and 5Ј-AGTGGTCCAATCTGCAGGTG-3Ј, respectively.…”

“…Interestingly, a recent report proposed that intermolecular electron transfer from the adjacent reductase domain to the oxygenase domain occurs in the homodimer of iNOS (24). Previous work in this laboratory suggested that basic amino acids such as Lys and Arg on the proximal surface of microsomal P450s are important for the interaction between the reductase and P450 for efficient electron transfer to occur (25,26). It is possible that similar interactions are required for electron transfer to occur in the homodimer of NOS, particularly in view of the structural rearrangement which probably occurs at the reductase domain-heme domain interface on CaM binding.…”

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras

“…The uncoupling process can lead to a loss of NADPH redox equivalents as high as 99% in human cytochrome P450 1A2 (Mayuzumi et al, 1993) or 16% for 3A4 (Perret and Pompon, 1998) and it can be seen as a wasteful process. These high uncoupling levels, together with the need of a reductase, are a considerable disadvantage in the use of human enzymes as biocatalysts.…”