Alpha-secondary Kinetic Isotope Effects Research Articles

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

The origin of hydrogen atoms during fatty acid biosynthesis in Fusarium lateritium has been quantified by isotope tracking close to natural abundance. Methyl linoleate was isolated from F. lateritium grown in natural abundance medium or in medium slightly enriched with labeled water, glucose, or acetate, and the (2)H incorporation was determined by quantitative (2)H-{(1)H} NMR in isotropic and chiral oriented solvents. Thus, the individual ((2)H/(1)H)(i) ratio at each pro-R and pro-S hydrogen position of the CH(2) groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (a(ij)) that characterize the specific source of each hydrogen atom to be related to the nonexchangeable hydrogen atoms in glucose and to the medium water. In turn, these can be related to the stereoselectivity that operates during the introduction or removal of hydrogens along the fatty acid chain. First, at even CH(2) the pro-S hydrogen comes only from water by protonation, whereas the pro-R hydrogen is introduced partly via acetate but principally from water. Second, the nonexchangeable hydrogens of glucose (positions H-6,6 and H-1) are shown to be introduced to the odd CH(2) via the NAD(P)H pool used by both reductases involved in the elongation steps of the fatty acid chain. Third, it is proved that hydrogens removed at sites 9,10 and 12,13 during desaturation by Delta(9)- and Delta(12)-desaturases are pro-R, and that during these desaturation steps alpha-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.

Mutagenesis of Morphinone Reductase Induces Multiple Reactive Configurations and Identifies Potential Ambiguity in Kinetic Analysis of Enzyme Tunneling Mechanisms

We have identified multiple reactive configurations (MRCs) of an enzyme-coenzyme complex that have measurably different kinetic properties. In the complex formed between morphinone reductase (MR) and the NADH analogue 1,4,5,6-tetrahydro-NADH (NADH4) the nicotinamide moiety is restrained close to the FMN isoalloxazine ring by hydrogen bonds from Asn-189 and His-186 as determined from the X-ray crystal structure. Molecular dynamic simulations indicate that removal of one of these hydrogen bonds in the N189A MR mutant allows the nicotinamide moiety to occupy a region of configurational space not accessible in wild-type enzyme. Using stopped-flow spectroscopy, we show that reduction of the FMN cofactor by NADH in N189A MR is multiphasic, identifying at least four different reactive configurations of the MR-NADH complex. This contrasts with wild-type MR in which hydride transfer occurs by environmentally coupled tunneling in a single kinetic phase [Pudney et al. J. Am. Chem. Soc. 2006, 128, 14053-14058]. Values for primary and alpha-secondary kinetic isotope effects, and their temperature dependence, for three of the kinetic phases in the N189A MR are consistent with hydride transfer by tunneling. Our analysis enables derivation of mechanistic information concerning different reactive configurations of the same enzyme-coenzyme complex using ensemble stopped-flow methods. Implications for the interpretation from kinetic data of tunneling mechanisms in enzymes are discussed.

Probing the Chemical Steps of Nitroalkane Oxidation Catalyzed by 2-Nitropropane Dioxygenase with Solvent Viscosity, pH, and Substrate Kinetic Isotope Effects

Among the enzymes that catalyze the oxidative denitrification of nitroalkanes to carbonyl compounds, 2-nitropropane dioxygenase is the only one known to effectively utilize both the neutral and anionic (nitronate) forms of the substrate. A recent study has established that the catalytic pathway is common to both types of substrates, except for the initial removal of a proton from the carbon of the neutral substrates [Francis, K., Russell, B., and Gadda, G. (2005) J. Biol. Chem. 280, 5195-5204]. In the present study, the mechanistic properties of the enzyme have been investigated with solvent viscosity, pH, and kinetic isotope effects. With nitroethane or ethylnitronate, the kcat/Km and kcat values were independent of solvent viscosity, consistent with the substrate and product binding to the enzyme in rapid equilibrium. The abstraction of the proton from the alpha carbon of neutral substrates was investigated by measuring the pH dependence of the D(kcat/KNE) value with 1,1-[2H2]-nitroethane. The formation of the enzyme-bound flavosemiquinone formed during catalysis was examined by determining the pH dependence of the kcat/Km values with ethylnitronate and nitroethane and the inhibition by m-nitrobenzoate. Finally, alpha-secondary kinetic isotope effects with 1-[2H]-ethylnitronate were used to propose a non-oxidative tautomerization pathway, in which the enzyme catalyzes the interconversion of nitroalkanes between their anionic and neutral forms. The data presented suggest that enzymatic turnover of 2-nitropropane dioxygenase with neutral substrates is limited by the cleavage of the substrate CH bond at low pH, whereas that with anionic substrates is limited by the non-oxidative tautomerization of ethylnitroante to nitroethane at high pH.

α-Secondary Isotope Effects as Probes of “Tunneling-Ready” Configurations in Enzymatic H-Tunneling: Insight from Environmentally Coupled Tunneling Models

Using alpha-secondary kinetic isotope effects (2 degrees KIEs) in conjunction with primary (1 degrees ) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2 degrees KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2 degrees KIEs, unlike 1 degrees KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2 degrees KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1 degrees KIE. The work emphasizes the benefit of combining studies of 1 degrees and 2 degrees KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.

Stereoselective Hydrogen Abstraction by Galactose Oxidase

The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.

Kinetic Isotope Effects and Transition State Geometries. A Theoretical Investigation of E2 Model Systems.

Ab initio calculations at the MP2/6-31+G level have been performed on E2 model systems to investigate whether differences in kinetic isotope effects correlate with changes in transition state geometries. By combining various nucleophiles (NH(2)(-), OH(-), F(-), PH(2)(-), SH(-), Cl(-)) and leaving groups (NH(3), Br(-), Cl(-), F(-), SH(-)) for reactions of the type Nu(-) + CH(3)CH(2)X, a large diversity of transition structures from reactant-like to product-like are generated. For each reaction one primary and two different alpha-secondary kinetic isotope effects are calculated. The primary kinetic isotope effects depend strongly on the nucleophilic placement in the periodic system, which mainly is due to differences in equilibrium isotope effects. When this effect is subtracted, the primary kinetic isotope effects display the expected maximum for symmetric transition structures, although the maximum is broad. The secondary kinetic isotope effects associated with the leaving group provide a qualitative correlation with the hybridization at the carbon, but the corresponding effects at the carbon where the hydrogen abstraction takes place is uncorrelated with the transition state geometry.

Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase.

The interaction of Lactobacillus casei thymidylate synthase (TS) with 10-propargyl-5,8-dideazafolate (NPQ) in the presence of 2'-deoxyuridylate (dUMP) has been investigated. After formation of a rapidly reversible dUMP-NPQ-enzyme complex, a slow isomerization occurs to provide a ternary complex that can be isolated on nitrocellulose membranes or by gel filtration. Unusual features of the isolable complex are the slow rate by which it is formed (t1/2 = 0.88 h) and the slow rate at which it dissociates (t1/2 = 26.5 h). The ternary complexes contain 2 mol of dUMP and 2 mol of NPQ bound per mol of dimeric enzyme. Ultraviolet difference spectra of the dUMP-NPQ-TS complex shows a high wavelength maximum that has been attributed to perturbations of the enzyme and/or ligand chromophores that occur upon binding. Data are presented that suggest that the formation of the isolable ternary complex involves nucleophilic attack by a catalytic thiol group of the enzyme to the 6-position of dUMP. Evidence for this is as follows: first, there is a decrease in the absorbance of the pyrimidine chromophore at 265 nm that occurs at the same rate as the formation of the isolable complex; second, using [6-3H]dUMP there is a large, inverse alpha-secondary kinetic isotope effect (kappa H/kappa T = 0.83) upon formation of the complex that is in accord with sp2 to sp3 rehybridization of the 6-carbon of the heterocycle. Treatment of the complex with sodium dodecyl sulfate (NaDodSO4) results in the dissociation of both ligands in an unmodified form, which is consistent with proposed structure of the complex. Isolable ternary complexes are also formed when the enzyme is incubated with 5-fluoro-2'-deoxyuridylate (FdUMP) and NPQ. Interestingly, the dissociation of FdUMP from these complexes is biphasic, with one-half of the bound nucleotide dissociating at an exceedingly slow rate (t1/2 congruent to 100 h). The findings are discussed with relationship to the possible use of NPQ as an anticancer agent.