Prochiral selectivity and intramolecular isotope effects in the cytochrome P-450 catalyzed .omega.-hydroxylation of cumene

• Published in: Biochemistry (Easton) Vol. 25; no. 23; pp. 7336 - 7343
• Main Authors: Sugiyama, Katsumi, Trager, William F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18-11-1986
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Benzene Derivatives - chemical synthesis; Benzene Derivatives - metabolism; Biological and medical sciences; Carbon Isotopes; cumene; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; Deuterium; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; Hydroxylation; Isotope Labeling - methods; Kinetics; Male; Mass Spectrometry; Mathematics; Microsomes, Liver - metabolism; Optical Rotation; Oxidoreductases; Rats; Rats, Inbred Strains; Stereoselectivity; Vertebrata; Mammalia; Isotope effect; Hydroxylation; Rat; Enzyme; Cytochrome P450; Liver; Rodentia; Stereochemistry; Microsome
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00371a015

A kinetic model is presented from which steady-state equations are derived that describe the intramolecular competition for the enzymatically mediated hydroxylation of two like groupings of a prochiral substrate. The observed isotope effect in such a system if one of the groupings is isotopically labeled is shown to be a function of three parameters: the equilibrium constant for the catalytically sensitive orientations of the two prochiral groupings at the active site, the intrinsic isotope effect associated with the bond-breaking step, and the relative rates of bond breaking vs. enzyme-substrate dissociation. The expected isotope effects associated with the omega-hydroxylation of racemic, (R)-, and (S)-2-phenylpropane-1,1,1-d3 and the product stereoselectivity associated with the omega-hydroxylation of (R)- and (S)-[1-13C]-2-phenylpropane were determined with microsomal preparations (cytochrome P-450) from untreated and phenobarbital- and beta-naphthoflavone-pretreated male Sprague-Dawley rats. The data from these experiments allow the observed isotope effect to be evaluated in terms of its component parts, i.e., expected isotope effects, product stereoselectivity, and equilibrium constant. These data further suggest that the intramolecular isotope effect is consistent with a hydrogen abstraction recombination mechanism and is largely dependent upon the chemical nature of the porphyrin-Fe-oxene complex but independent of specific apoprotein structure, product stereoselectivity is primarily dependent upon apoprotein structure, and product stereoselectivity is a good measure of the equilibrium constant and both parameters are dependent upon the chirality of the active site.