Quantum mechanics/molecular mechanics modeling of drug metabolism:Mexiletine N-hydroxylation by cytochrome P450 1A2

The mechanism of cytochrome P450­(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (<i>R</i>)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM­(B3LYP-D)/MM­(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism

Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme

Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment

Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O_2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O_2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O_2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs

Experimental approaches to evaluate activities of cytochromes P450 3A

Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant

Cheek Tooth Morphology and Ancient Mitochondrial DNA of Late Pleistocene Horses from the Western Interior of North America: Implications for the Taxonomy of North American Late Pleistocene Equus

Horses were a dominant component of North American Pleistocene land mammal communities and their remains are well represented in the fossil record. Despite the abundant material available for study, there is still considerable disagreement over the number of species of Equus that inhabited the different regions of the continent and on their taxonomic nomenclature. In this study, we investigated cheek tooth morphology and ancient mtDNA of late Pleistocene Equus specimens from the Western Interior of North America, with the objective of clarifying the species that lived in this region prior to the end-Pleistocene extinction. Based on the morphological and molecular data analyzed, a caballine (Equus ferus) and a non-caballine (E. conversidens) species were identified from different localities across most of the Western Interior. A second non-caballine species (E. cedralensis) was recognized from southern localities based exclusively on the morphological analyses of the cheek teeth. Notably the separation into caballine and non-caballine species was observed in the Bayesian phylogenetic analysis of ancient mtDNA as well as in the geometric morphometric analyses of the upper and lower premolars. Teeth morphologically identified as E. conversidens that yielded ancient mtDNA fall within the New World stilt-legged clade recognized in previous studies and this is the name we apply to this group. Geographic variation in morphology in the caballine species is indicated by statistically different occlusal enamel patterns in the specimens from Bluefish Caves, Yukon Territory, relative to the specimens from the other geographic regions. Whether this represents ecomorphological variation and/or a certain degree of geographic and genetic isolation of these Arctic populations requires further study

Disease concepts and treatment by tribal healers of an Amazonian forest culture

<p>Abstract</p> <p>Background</p> <p>The extensive medicinal plant knowledge of Amazonian tribal peoples is widely recognized in the scientific literature and celebrated in popular lore. Despite this broad interest, the ethnomedical systems and knowledge of disease which guide indigenous utilization of botanical diversity for healing remain poorly characterized and understood. No study, to our knowledge, has attempted to directly examine patterns of actual disease recognition and treatment by healers of an Amazonian indigenous culture.</p> <p>Methods</p> <p>The establishment of traditional medicine clinics, operated and directed by elder tribal shamans in two remote Trio villages of the Suriname rainforest, presented a unique investigational opportunity. Quantitative analysis of clinic records from both villages permitted examination of diseases treated over a continuous period of four years. Cross-cultural comparative translations were articulated of recorded disease conditions through ethnographic interviews of elder Trio shamans and a comprehensive atlas of indigenous anatomical nomenclature was developed.</p> <p>Results</p> <p>20,337 patient visits within the period 2000 to 2004 were analyzed. 75 disease conditions and 127 anatomical terms are presented. Trio concepts of disease and medical practices are broadly examined within the present and historical state of their culture.</p> <p>Conclusion</p> <p>The findings of this investigation support the presence of a comprehensive and highly formalized ethnomedical institution within Trio culture with attendant health policy and conservation implications.</p