Pre-steady-state kinetics of incorporation of dCTP and dATP opposite site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua), in contrast to dCTP insertion opposite G, were examined as well as extension beyond the lesion using the replicative enzymes bacteriophage polymerase T7 exo- (T7-) and HIV-1 reverse transcriptase (RT). These results were compared to previous findings for Escherichia coli repair polymerases I (KF-) and II (pol II-) exo- [Lowe, L. G., & Guengerich, F. P. (1996) Biochemistry 35, 9840-9849]. HIV-1 RT showed a very high preference for insertion of dATP opposite 8-oxoGua, followed by pol II-, T7-, and KF-. Steady-state assays showed k(cat) consistently lower than pre-steady-state polymerization rates (k(p)) for insertion of dCTP opposite G or 8-oxoGua and insertion of dATP opposite 8-oxoGua. Pre-steady-state kinetic curves for the addition of dCTP opposite 8-oxoGua or G by KF-, pol II-, and T7- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of dATP opposite 8-oxoGua by these polymerases did not display burst kinetics. With HIV-1 RT, addition of dATP opposite 8-oxoGua displayed burst kinetics while addition of dCTP did not. Analyses of the chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7-; HIV- RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher Kd and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K(d,app)dATP values for KF-, pol II-, and T7- incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K(d)dCTP. Likewise, the K(d,app)dCTP for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K(d)dATP. The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua x A pair; extension of 8-oxoGua x C was severely impaired, whereas the replicative enzymes (T7- and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua x A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent Kd, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.

The majority of pre-steady-state kinetic investigations with HIV-1 reverse transcriptase (HIV-1 RT) have reported substoichiometric bursts (30-50%) of product formation in the initial reaction cycle. By using quantitative amino acid analysis, we have revised the extinction coefficient of the HIV-1 RT heterodimer and show that normal nucleotide incorporation (canonical four bases) proceeds with quantitative bursts in the first cycle. We have also modeled our previous results with this polymerase, including four situations with 8-oxo-7,8-dihydroguanine (8-oxoGua) moieties in which substoichiometric bursts (2-35%) were observed even after the correction of enzyme concentration by amino acid analysis. These include insertion of dATP opposite template 8-oxoGua, insertion of (deoxy) 8-oxoGua 5'-triphosphate opposite template C, and extension of primers beyond 8-oxoGua-A and 8-oxoGua-C pairs. The "minimal" polymerase mechanism and three others were evaluated using KINSIM and FITSIM methods. The latter three mechanisms involve a conformationally distinct, inactive polymerase-DNA-dNTP complex in equilibrium with the initial ternary complex and a conformationally distinct complex leading to phosphodiester bond formation. All three of the modified mechanisms fit the observed reaction results, but the minimal mechanism did not. Nonfunctional binary complexes (enzyme-DNA) are an alternate explanation (to ternary complexes) in some cases. Finally, DNA trapping experiments indicate that enzyme does not dissociate from the 8-oxoGua-containing DNA substrate prior to phosphodiester bond formation. We conclude that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with modified systems are well-described by the existence of nonproductive ternary complexes, which can isomerize to productive complexes.

Cytochrome P450 (P450) enzymes include a family of related enzymes that are involved in metabolism of vitamins, steroids, fatty acids, and other chemicals. This review presents a brief historical overview of the discovery and characterization of P450 enzymes extending from intermediary metabolism to the fields of drug metabolism and toxicology. Introductions to P450 enzyme structure and function are also presented. The goals of this review are to 1) provide an introduction to a few of the many aspects of P450 research relating to humans, 2) introduce additional ways of thinking about metabolism, 3) provide some basic examples of P450 enzymology, and 4) provide applications to topics widely taught in undergraduate courses in biochemistry.Keywords: Cytochrome P450 enzyme, drug metabolism, chemical toxicity, P450 mechanism.Cytochrome P450 (P450) 1 enzymes are a family of heme-containing proteins found from bacteria to human. In mammals, the enzymes are membrane-bound (usually in the endoplasmic reticulum, although the seven in Families 11, 24, and 27 are found in mitochondria; Table I). P450s serve a variety of functions including steroid, fatty acid, eicosanoid, and vitamin A and D metabolism as well as metabolism of foreign compounds including natural products, pharmaceuticals, and carcinogens. P450s are found in every tissue except skeletal muscle and red blood cells [1]. With the completion of the human genome sequence project, there appear to be 57 distinct P450 genes in humans [2]. Of these, about 13 are "orphan" P450s for which the metabolic function is not yet known [3].There is wide interest in P450s due to applications in a variety of fields including biochemistry, biotechnology, chemistry, environmental sciences, enzymology, microbiology, physiology, pharmacology, plant sciences, and toxicology. Indeed, in the last decade alone, there were more than 1000 publications per year relating to P450s. This review will highlight the roles of human P450s in drug metabolism and chemical toxicity and applications to undergraduate studies. References to more in-depth reviews are also provided. DISCOVERY AND PURIFICATIONThe history of P450 can be traced back to questions related to the metabolism of steroids, drugs, and carcinogens in the 1940s. Early studies of difficult alkyl oxidations of amino acids and fatty acids, such as oxidation at the terminal methyl group of a fatty acid, allowed for the isolation of cell-free extracts capable of performing NAD(P)Hdependent oxidation reactions [4]. The "mixed-function oxidase" stoichiometry.that is now recognized as the basic reaction catalyzed by a P450 was not immediately obvious. In particular, the requirement for a reduced pyridine nucleotide to achieve an overall oxidation was surprising at the time. The concept of mixed-function oxidation was developed by O. Hayaishi et al. in Japan [5] and H. Mason in the U.S [6]. Subsequently, such reactions were shown to occur with steroids and xenobiotics, particularly drugs (J. Axelrod and B. Brodie et al.) [7,8] and carcinogen...

Recent studies have demonstrated that two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1, 2-dithiole-3-thione (D3T), and sulforaphane (SF), an isothiocyanate, are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes (P450s) involved in xenobiotic metabolism. We examined P450 inhibition by the two compounds and compared two OPZ metabolites (OPZ M(3) and M(8)) and D3T using human P450s expressed in Escherichia coli membranes. OPZ was a more potent inhibitor than D3T or SF, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 approximately 1B1 > 2E1. OPZ M(3) also inhibited P450s 1A2, 1A1, 1B1, and 3A4 but not more effectively than OPZ. OPZ M(8) was not inhibitory. OPZ behaved as a competitive inhibitor of P450 1A2, with a K(i) of 1.5 microM. Incubation of P450 1A2 with OPZ and NADPH led to a first-order loss of the P450 spectrum, and the loss was not blocked by glutathione. No such time-dependent loss of P450 was seen with P450 1A2 and D3T, P450 1A2 and OPZ M(3), P450 1A2 and SF, P450 3A4 and OPZ, P450 3A4 and D3T, P450 2E1 and OPZ, or P450 2E1 and D3T. The time- and concentration-dependent loss of P450 1A2 activity in the presence of OPZ was characterized with a K(i) of 9 microM and a k(inactivation) of 0.19 min(-)(1). The activation of 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline (MeIQ) in an E. coli lac-based mutagenicity tester system containing functional human P450 1A2 was inhibited by OPZ (IC(50) < 1 microM) but not appreciably by 40 microM D3T. Our results indicate that OPZ is a competitive and mechanism-based inhibitor of P450 1A2, and the extent of this inhibition is significantly greater than that of other chemoprotective chemicals with P450 1A2 or other human P450s.

Cytochrome P450 enzymes (CYPs) represent an important enzyme superfamily involved in metabolism of many endogenous and exogenous small molecules. CYP2D6 is responsible for ∼15% of CYP-mediated drug metabolism and exhibits large phenotypic diversity within CYPs with over 100 different allelic variants. Many of these variants lead to functional changes in enzyme activity and substrate selectivity. Herein, a molecular dynamics comparative analysis of four different variants of CYP2D6 was performed. The comparative analysis included simulations with and without SCH 66712, a ligand that is also a mechanism-based inactivator, in order to investigate the possible structural basis of CYP2D6 inactivation. Analysis of protein stability highlighted significantly altered flexibility in both proximal and distal residues from the variant residues. In the absence of SCH 66712, *34, *17-2, and *17-3 displayed more flexibility than *1, and *53 displayed more rigidity. SCH 66712 binding reversed flexibility in *17-2 and *17-3, through *53 remained largely rigid. Throughout simulations with docked SCH 66712, ligand orientation within the heme-binding pocket was consistent with previously identified sites of metabolism and measured binding energies. Subsequent tunnel analysis of substrate access, egress, and solvent channels displayed varied bottle-neck radii. Taken together, our results indicate that SCH 66712 should inactivate these allelic variants, although varied flexibility and substrate binding-pocket accessibility may alter its interaction abilities.