Differential roles of Glu318 and Thr319 in cytochrome P450 1A2 catalysis supported by NADPH-cytochrome P450 reductase and tert-butyl hydroperoxide.
The kinetic values for 7-ethoxycoumarin (7-EC) hydroxylation have been obtained in both the NADPH-cytochrome P450 reductase- and tert-butyl hydroperoxide (TBHP)-supported systems for several Glu318 and Thr319 mutants of cytochrome P450 1A2. The results with the reductase-supported system suggest that Glu318 is important for both substrate binding and catalysis, whereas Thr319 is critical for neither, although the size of the residue at position 319 influences catalytic activity. In contrast, neither Glu318 nor Thr319 appears to be important for catalytic turnover in the TBHP-supported system despite the fact that the size of the amino acid at position 319 affects the binding of TBHP and 7-EC in opposite manners. The roles of these two distal amino acids in the cytochrome P450 1A2-catalyzed oxidation of 7-EC therefore differ for the reactions supported by cytochrome P450 reductase and TBHP.
Hiroya
K
K
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Murakami
Y
Y
Shimizu
T
T
Hatano
M
M
Ortiz de Montellano
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013912:Threonine; D020122:tert-Butylhydroperoxide
D005971:Glutamates; D010545:Peroxides; D011994:Recombinant Proteins; D013912:Threonine; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; D020122:tert-Butylhydroperoxide; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase