Kinetic Deuterium Isotope Effects in Cytochrome P450 Reactions

Abstract

Cytochrome P450 (P450, CYP) research provides many opportunities for the application of kinetic isotope effect (KIE) strategies. P450s collectively catalyze oxidations of more substrates than any other group of enzymes, and C-H bond cleavage is a major feature in a large fraction of these reactions. The presence of a significant primary deuterium KIE is evidence that hydrogen abstraction is at least partially rate-limiting in the reactions, and this appears to be the case in many P450 reactions. The first report of a KIE in (P450-linked) drug metabolism appeared in 1961 (for morphine N-demethylation), and in a number of cases it has been possible to modulate the in vivo metabolism or toxicity of chemicals by deuterium substitution. A number of efforts are in progress to utilize deuterium substitution to alter the metabolism of drugs in an advantageous manner.

A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450‐Catalyzed Substrate Conversion

“…Thisi si nl ine with previous studies in which substantially larger KIEs were observed for aliphatic than for aromatic hydroxylation by P450s. [30] The increasein4'-OH-MF formationa fter deuteration may be well explained by metabolic switching resulting from the strongly decreased 3'-hydroxylation. Thus, our kinetic isotope effect measurements for these reactionss uggestt hat rapid interconversion between catalytically active poses for 3'-OH-MF and 4'-OH-MF formation is possible and accordingly,that k i and k Ài values for binding-poseinterchange and/or k b and k u values are probably highert han k p for the corresponding product formations.…”

“…13 ]mefenamic acid, so that eventual effects of these side products on the reactions were applicable to both labelled and unlabeled mefenamic acid to the same extent. [30] All incubations were performed with 100 nm P450 in 100 mm potassium phosphate buffer (pH 7.4) supplemented with 5mm MgCl 2 and 2mm EDTA. To tal substrate concentrations of the mixture of labelled and unlabeled mefenamic acid were 75 and 750 mm.R eaction mixtures (total volume 100 mL) were pre-warmed at the incubation temperature for 10 min, before initiating the reaction by addition of aN ADPH regenerating system (final concentrations of 0.5 mm NADPH, 10 mm glucose 6-phosphate, and 0.4 unit mL À1 glucose-6-phosphate dehydrogenase).…”

“…Which step is rate limiting appears to depend on the specific combination of P450 isoform and substrate involved in the hydroxylation reaction. [21][22][23][24][25][26][27][28][29][30] For severals ubstrates the rate-limiting nature of hydrogen abstraction has been demonstrated by the kinetic isotope effect (KIE) observed after deuterium substitution. [23,27,30] The regio-(and stereo-)selectivity and specific activity of cyto-chromeP 450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrater elativet o the heme, and the activation barrier of the regioselective oxidation reaction(s).…”

“…[21][22][23][24][25][26][27][28][29][30] For severals ubstrates the rate-limiting nature of hydrogen abstraction has been demonstrated by the kinetic isotope effect (KIE) observed after deuterium substitution. [23,27,30] The regio-(and stereo-)selectivity and specific activity of cyto-chromeP 450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrater elativet o the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative bindingf ree energy (DDG bind )ofthe catalytically active substrate-binding poses, and the probability of the substrate to adopt at ransition-state geometry.A ne stablished experimental method to measure activation energies of enzymat-ic reactions is the analysis of reactionr ate constants at different temperatures and the constructiono fA rrhenius plots.…”

Polarimetric Research of Pharmaceutical Substances in Aqueous Solutions With Different Water Isotopologues Ratio

“…The second direction refers to the use of deuterium depleted water as an adjuvant in the treatment of cancer [19,20]. The result of the change in D/H ratio is manifested in the form of kinetic isotopic effect [21][22][23][24], which is characterized by a change in the rate of absorption, distribution, biotransformation, and excretion of the medicines. Development of methodological approaches to drugs quality control based on water O) mutarotation of carbohydrates had the different kinetic mechanisms.…”