Intramolecular Isotope Effects for Benzylic Hydroxylation of Isomeric Xylenes and 4,4‘-Dimethylbiphenyl by Cytochrome P450:  Relationship between Distance of Methyl Groups and Masking of the Intrinsic Isotope Effect

• Published in: Biochemistry (Easton) Vol. 36; no. 23; pp. 7136 - 7143
• Main Authors: Iyer, Krishna R, Jones, Jeffrey P, Darbyshire, John F, Trager, William F
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.06.1997
• Subjects: Animals; Benzene - metabolism; Biphenyl Compounds - metabolism; Cytochrome P-450 CYP2B1 - metabolism; Cytochrome P-450 Enzyme System - metabolism; Deuterium; Hydrogen Bonding; Hydroxylation; Isomerism; Kinetics; Male; Methylation; Microsomes, Liver - enzymology; Models, Chemical; Rats; Rats, Sprague-Dawley; Xylenes - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi962810m

Intramolecular isotope effects associated with the benzylic hydroxylation of a series of selectively deuterated isomeric xylenes and 4,4‘-dimethylbiphenyl as catalyzed by various rat liver microsomal preparations and CYP2B1 were determined. Substrate analogs in which each methyl group contained either one (d 2 substrates) or two (d 4 substrates) deuterium atoms were used to determine the intrinsic isotope effect for the reaction. Specific values of the individual primary (P) and secondary isotope effects (S) were determined. P ranged from a low of 5.32 ± 0.48 to a high of 7.57 ± 0.42 depending upon the specific cytochrome P450 preparation used for catalysis. S had an average value of 1.03. The d 3 substrates allowed exploration of the effect of distance on the magnitude of the observed isotope effect. The results indicate that the distance of 6.62 Å that separates the carbon atoms of the para methyl groups of p-xylene is insufficient to suppress (mask) the intrinsic isotope effect for benzylic hydroxylation by all of the enzyme preparations examined. Conversely, a distance of 11.05 Å, the minimal separation between the carbon atoms of the para methyl groups of p,p‘-dimethylbiphenyl, is large enough to almost completely mask the intrinsic isotope effect for benzylic hydroxylation by the same set of enzymes.