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664888 
Journal Article 
Oxidation kinetics of ethanol by human cytochrome P450 2E1. Rate-limiting product release accounts for effects of isotopic substitution and cytochrome b5 on steady-state kinetics 
Bell, LC; Guengerich, FP 
1997 
Yes 
Journal of Biological Chemistry
ISSN: 0021-9258
EISSN: 1083-351X 
272 
47 
29643-29651 
English 
9368031 
WOS:A1997YG64700041 
A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of 5 on Km (DK = DKmm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k > 1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. 
Biochemistry; Amino acids; Peptides; Proteins; Bile pigments; Porphyrins; Coenzymes; Comparative study; Enzymes; Enzymes physiology