Browsing by Subject "isotope effects"
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Item MECHANISM OF OXYGEN ACTIVATION AND HYDROXYLATION BY THE AROMATIC AMINO ACID HYDROXYLASES(2010-07-14) Pavon, Jorge A.The aromatic amino acid hydroxylases phenylalanine hydroxylase (PheH), tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) utilize tetrahydropterin and molecular oxygen to catalyze aromatic hydroxylation. All three enzymes have similar active sites and contain an iron atom facially coordinated by two histidines and a glutamate. The three enzymes also catalyze the benzylic hydroxylation of 4- methylphenylalanine. The intrinsic primary and ?-secondary isotope effects for benzylic hydroxylation and their temperature dependences are nearly identical for the three enzymes, suggesting that the transition states, the tunneling contributions and the reactivities of the iron centers are the same. When molecular oxygen and the tetrahydropterin are replaced by hydrogen peroxide (H2O2), these enzymes catalyze the hydroxylation of phenylalanine to form tyrosine and meta-tyrosine with nearly identical second order rate constants. When the H2O2-dependent reaction is carried out with cyclohexylalanine or 4-methylphenylalanine, the products are 4-HO-cyclohexylalanine and 4-hydroxymethylphenylalanine, respectively. These experiments provide further evidence that the intrinsic reactivities of the iron centers in these enzymes are the same. Wild-type PheH and the uncoupled mutant protein V379D exhibit normal and inverse isotope effects, respectively, with deuterated phenylalanines. When the reaction is monitored by stopped-flow absorbance spectroscopy, three steps are visible. The first step is the reversible binding of O2, the second step is 5-7 fold faster than the turnover number, setting a limiting value for the rate constant for O2 activation, and the last step is non-enzymatic. There is no burst in the pre-steady state formation of tyrosine. These results are consistent with formation of the new C-O bond to form tyrosine as the ratelimiting step of the reaction. The reaction of TrpH with both tryptophan and phenylalanine was studied by stopped-flow absorbance spectroscopy and rapid-quench product analysis. With either amino acid as substrate, four steps can be distinguished. The first step is the reversible binding of O2 to the Fe(II) center; this results in an absorbance signature with a maximum at 420 nm. This O2 complex decays with a rate constant that is 18-22 fold faster than the turnover number with either amino acid, setting a the lower limit for the rate constant for O2 activation. The rate constant for the third step agrees well with the pre-steady state of formation of 5-hydroxytryptophan or tyrosine from rapid-quench product analysis. The rate constant for the fourth step agrees well with the turnover number. Overall, these results show that O2 activation is fast and turnover with each amino acid is limited by hydroxylation and release of a product, with the former step being about 4-fold faster than the latter.Item Observation and Nature of Non-statistical Dynamic Effects in Ordinary Organic Reactions(2012-10-17) Quijano, Larisa Mae Mangaliman 1984-Statistical models like Transition State Theory (TST) and Rice-Ramsperger-Kassel-Marcus (RRKM) Theory have generally been successful in predicting the rates and selectivities of chemical reactions. However, these statistical models can fail to explain experimental results of ordinary organic reactions. For these reactions, consideration of nonstatistical dynamic effects or the detailed motion and momenta of the atoms is necessary to account for the experimental observations. Dynamic effects have been found to be important in a growing number of reactions and the nature of these effects can be varied. One of the most interesting reactions investigated is the ozonolysis of vinyl ethers. Ozonolysis of a homologous series of vinyl ethers in solution exhibit experimental product ratios wherein the selectivity among cleavage pathways increases with the size of the alkyl group to an extent that is far less than RRKM theory would predict. Trajectory studies account for the observed selectivities and support a mechanism involving a competition between cleavage of the primary ozonide and intramolecular vibrational energy redistribution. A recent theoretical study from our group predicted that a highly asynchronous organocatalytic Diels-Alder (DA) reaction, which is concerted in the potential energy surface, is stepwise in the free energy surface. Kinetic isotope effects (KIEs) were measured for three DA reactions. We envision that the entropic barrier may have several experimental consequences such as unusual isotope effects due to extensive recrossing. Preliminary results for the organocatalytic reaction show an intramolecular KIE close to unity that cannot be reconciled with statistical theories. This is in contrast with Lewis-acid catalyzed and thermal DA reactions, which exhibit substantial "normal" intramolecular KIEs that are in accord with TST predictions. Finally, the Baeyer-Villiger oxidation of cylohexanone in water was investigated. KIEs were measured for the oxidation of cyclohexanone with peracetic acid and trifluoroperacetic acid. When using peracetic acid as the oxidant, the alkyl migration was determined to be the rate-determining step based on significant intermolecular KIEs on the carbonyl and alpha-methylene carbons. A change in the rate-determining step is seen when trifluoroperacetic acid is used. Only the carbonyl carbon exhibits a significant isotope effect. Theoretical predictions provide an experimental picture of the transition states and qualitatively support these conclusions.Item Structure and function of circadian clock proteins and deuterium isotope effects in nucleic acid hydrogen bonds(Texas A&M University, 2005-08-29) Vakonakis, IoannisCircadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.Item The chemical mechanisms of flavin-dependent amine oxidases and the plasticity of the two-his one-carboxylate facial triad in tyrosine hydroxylase(2009-05-15) Ralph, Erik C.Despite a number of kinetic and spectroscopic studies, the chemical mechanisms of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were probed using a combination of pH and primary deuterium, solvent, and 15N kinetic isotope effects. Slow substrates were chosen for these studies; MTOX was characterized with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX and for alanine oxidation by TMO, respectively, independent of the substrate concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no intermediate flavin species with both enzyme-substrate systems. Furthermore, the magnitudes of the 15N kinetic isotope effects observed with both systems suggest that nitrogen rehybridization and C-H bond cleavage are concerted. These results are consistent with both enzymes utilizing a hydride transfer mechanism for amine oxidation. The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or histidine. The resulting proteins were characterized for metal content, catalytic activity, and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted relative to that of the wild-type enzyme, consistent with the change in the metal ligand. In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results provide a reference point for further structural studies of TyrH and the other aromatic amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding motif.