Large Deuterium Kinetic Isotope Effect Research Articles

Primary Deuterium Kinetic Isotope Effects: A Probe for the Origin of the Rate Acceleration for Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase.

Large primary deuterium kinetic isotope effects (1° DKIEs) on enzyme-catalyzed hydride transfer may be observed when the transferred hydride tunnels through the energy barrier. The following 1° DKIEs on kcat/Km and relative reaction driving force are reported for wild-type and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reactions of NADL (L = H, D): wild-type GPDH, ΔΔG⧧ = 0 kcal/mol, 1° DKIE = 1.5; N270A, 5.6 kcal/mol, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1° DKIEs were observed on kcat. The narrow range of 1° DKIEs (2.4–3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1° DKIEs on rate-determining hydride transfer. Evidence is presented that the intrinsic DKIE on wild-type GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1° DKIEs (2.4–2.9) on (kcat/KGA, M–1 s–1) was reported for dianion-activated hydride transfer from NADL to glycolaldehyde (GA) [Reyes, A. C.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc.2016, 138, 14526–14529]. These 1° DKIEs are much smaller than those observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum mechanical tunneling. These results support the conclusion that the rate acceleration for GPDH-catalyzed reactions is due to the stabilization of the transition state for hydride transfer by interactions with the protein catalyst. The small 1° DKIEs reported for mutant GPDH-catalyzed and for wild-type dianion-activated reactions are inconsistent with a model where the dianion binding energy is utilized in the stabilization of a tunneling ready state.

Proton‐Coupled Electron Transfer in Soybean Lipoxygenase: Hydrogen Tunneling, Electrostatics, and Conformational Motions

The enzyme soybean lipoxygenase (SLO) serves as a prototype for hydrogen tunneling reactions because of its unusually large deuterium kinetic isotope effect (KIE) of 80 for the wild‐type enzyme and over 500 for a double mutant. The proton‐coupled electron transfer (PCET) reaction catalyzed by SLO has been studied with a vibronically nonadiabatic PCET theory that includes the quantum mechanical effects of the electrons and the transferring proton, as well as the proton donor‐acceptor and solvent motions. The calculations reproduced the experimentally observed magnitude and temperature dependence of the KIE for wild‐type SLO and a series of single mutants. The large KIE was found to arise from the small overlap between the reactant and product proton vibrational wave functions. Theoretical modeling of the double mutant illustrated that the colossal KIE of over 500 arises from a combination of a non‐optimal active site conformation corresponding to longer equilibrium donor‐acceptor distances and reduced sampling of shorter donor‐acceptor distances. Recent quantum mechanical/molecular mechanical (QM/MM) free energy simulations using a finite temperature string method combined with umbrella sampling have provided additional insights about the conformational motions, as well as the significance of electrostatics in this enzymatic reaction. These calculations highlight the critical roles of hydrogen tunneling, electrostatics, and protein conformational motions in enzyme catalysis.Support or Funding InformationNational Institutes of Health Grant GM056207This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Primary deuterium kinetic isotope effects prolong drug release and polymer biodegradation in a drug delivery system

We have developed a chemically-controlled drug delivery system in which a drug is covalently attached via a carbamate to hydrogel microspheres using a β-eliminative linker; rate-determining proton removal from a CH bond adjacent to an electron withdrawing group results in a β-elimination to cleave the carbamate and release the drug. After subcutaneous injection of the hydrogel-drug conjugate, the drug is slowly released into the systemic circulation and acquires an elimination t1/2,β that matches the t1/2 of linker cleavage. A similar β-eliminative linker with a slower cleavage rate is installed into crosslinks of the polymer to trigger gel degradation after drug release. We have now prepared β-eliminative linkers that contain deuterium in place of the hydrogen whose removal initiates cleavage. In vitro model systems of drug release and degelation show large primary deuterium kinetic isotope effects of kH/kD ~ 2.5 to 3.5. Using a deuterated linker to attach the peptide octreotide to hydrogel-microspheres, the in vivo t1/2,β of the drug was increased from ~1.5 to 4.5 weeks in the rat. Similarly, the in vivo time to biodegradation of hydrogels with deuterium-containing crosslinks could be extended ~2.5-fold compared to hydrogen-containing counterparts. Thus, the use of primary deuterium kinetic isotope effects in a single platform technology can control rates of β-elimination reactions in drug release and polymer biodegradation rates.

Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli

The enzyme QueF catalyzes a four-electron reduction of a nitrile group into an amine, the only reaction of this kind known in biology. In nature, QueF converts 7-cyano-7-deazaguanine (preQ0) into 7-aminomethyl-7-deazaguanine (preQ1) for the biosynthesis of the tRNA-inserted nucleoside queuosine. The proposed QueF mechanism involves a covalent thioimide adduct between preQ0 and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ1 in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ0 in a strongly exothermic process (ΔH = −80.3 kJ/mol; −TΔS = 37.9 kJ/mol, Kd = 39 nm) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ0 reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4–7-fold more slowly than trapping of the enzyme-bound preQ0 as covalent thioimide (1.63 s−1) and are thus mainly rate-limiting for the enzyme's kcat (=0.12 s−1). Kinetic studies combined with simulation reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ1. 7-Formyl-7-deazaguanine, a carbonyl analogue of the imine intermediate, was synthesized chemically and is shown to be recognized by QueF as weak ligand for binding (ΔH = −2.3 kJ/mol; −TΔS = −19.5 kJ/mol) but not as substrate for reduction or oxidation. A model of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data.

Switchover of the Mechanism between Electron Transfer and Hydrogen‐Atom Transfer for a Protonated Manganese(IV)–Oxo Complex by Changing Only the Reaction Temperature

AbstractHydroxylation of mesitylene by a nonheme manganese(IV)–oxo complex, [(N4Py)MnIV(O)]2+ (1), proceeds via one‐step hydrogen‐atom transfer (HAT) with a large deuterium kinetic isotope effect (KIE) of 3.2(3) at 293 K. In contrast, the same reaction with a triflic acid‐bound manganese(IV)‐oxo complex, [(N4Py)MnIV(O)]2+‐(HOTf)2 (2), proceeds via electron transfer (ET) with no KIE at 293 K. Interestingly, when the reaction temperature is lowered to less than 263 K in the reaction of 2, however, the mechanism changes again from ET to HAT with a large KIE of 2.9(3). Such a switchover of the reaction mechanism from ET to HAT is shown to occur by changing only temperature in the boundary region between ET and HAT pathways when the driving force of ET from toluene derivatives to 2 is around −0.5 eV. The present results provide a valuable and general guide to predict a switchover of the reaction mechanism from ET to the others, including HAT.

Switchover of the Mechanism between Electron Transfer and Hydrogen‐Atom Transfer for a Protonated Manganese(IV)–Oxo Complex by Changing Only the Reaction Temperature

Hydroxylation of mesitylene by a nonheme manganese(IV)-oxo complex, [(N4Py)Mn(IV) (O)](2+) (1), proceeds via one-step hydrogen-atom transfer (HAT) with a large deuterium kinetic isotope effect (KIE) of 3.2(3) at 293 K. In contrast, the same reaction with a triflic acid-bound manganese(IV)-oxo complex, [(N4Py)Mn(IV) (O)](2+) -(HOTf)2 (2), proceeds via electron transfer (ET) with no KIE at 293 K. Interestingly, when the reaction temperature is lowered to less than 263 K in the reaction of 2, however, the mechanism changes again from ET to HAT with a large KIE of 2.9(3). Such a switchover of the reaction mechanism from ET to HAT is shown to occur by changing only temperature in the boundary region between ET and HAT pathways when the driving force of ET from toluene derivatives to 2 is around -0.5 eV. The present results provide a valuable and general guide to predict a switchover of the reaction mechanism from ET to the others, including HAT.

Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH− or MnIIIOH−. Hydrogen abstraction by FeIIIOH− of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-2H2]18:2n-6 were almost temperature independent with an apparent D-KIE of ∼56 at 30°C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8–50°C), and the apparent D-KIE decreased linearly from ∼38 at 8°C to ∼20 at 50°C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor–acceptor distances, and to the metal ligands, which may not equalize the 0.7V-gap between the redox potentials of the free metals.

Kinetic and Mechanistic Studies of Carbon-to-Metal Hydrogen Atom Transfer Involving Os-Centered Radicals: Evidence for Tunneling

We have investigated the kinetics of novel carbon-to-metal hydrogen atom transfer reactions, in which homolytic cleavage of a C-H bond is accomplished by a single metal-centered radical. Time-resolved IR spectroscopic measurements revealed efficient hydrogen atom transfer from xanthene, 9,10-dihydroanthracene, and 1,4-cyclohexadiene to Cp(CO)2Os(•) and (η(5)-(i)Pr4C5H)(CO)2Os(•) radicals, formed by photoinduced homolysis of the corresponding osmium dimers. The rate constants for hydrogen abstraction from these hydrocarbons are in the range 1.5 × 10(5) M(-1) s(-1) to 1.7 × 10(7) M(-1) s(-1) at 25 °C. For the first time, kinetic isotope effects for carbon-to-metal hydrogen atom transfer were determined. Large primary deuterium kinetic isotope effects of 13.4 ± 1.0 and 16.8 ± 1.4 were observed for the hydrogen abstraction from xanthene to form Cp(CO)2OsH and (η(5)-(i)Pr4C5H)(CO)2OsH, respectively, at 25 °C. Temperature-dependent measurements of the kinetic isotope effects over a 60 °C temperature range were carried out to obtain the difference in activation energies (E(D) - E(H)) and the pre-exponential factor ratio (A(H)/A(D)). For hydrogen atom transfer from xanthene to (η(5)-(i)Pr4C5H)(CO)2Os(•), the (E(D) - E(H)) = 3.3 ± 0.2 kcal mol(-1) and A(H)/A(D) = 0.06 ± 0.02 values suggest a quantum mechanical tunneling mechanism.

ChemInform Abstract: Sequential Norrish Type II Photoelimination and Intramolecular Aldol Cyclization of α‐Diketones: Synthesis of Polyhydroxylated Cyclopentitols by Ring Contraction of Hexopyranose Carbohydrate Derivatives.

The excitation of the innermost carbonyl of nono-2,3-diulose derivatives by irradiation with visible-light initiates a sequential Norrish typeII photoelimination and aldol cyclization process that finally gives polyfunctionalized cyclopentitols. The rearrangement has been confirmed by the isolation of stable acyclic photoenol intermediates that can be independently cyclized by a thermal 5-(enolexo)-exo-trig uncatalyzed aldol reaction with high diastereoselectivity. In this last step, the large deuterium kinetic isotope effect found for the 1,5-hydrogen atom transfer seems to indicate that the aldol reaction runs through a concerted pericyclic mechanism. Owing to the ready availability of pyranose sugars of various configurations, this protocol has been used to study the influence of pyranose ring-substituents on the diastereoselectivity of the aldol cyclization reaction. In contrast with other pyranose ring contraction methodologies no transition-metal reagents are needed and the sequential rearrangement occurs simply by using visible light and moderate heating (0 to 60 degrees C).

Sequential Norrish Type II Photoelimination and Intramolecular Aldol Cyclization of α‐Diketones: Synthesis of Polyhydroxylated Cyclopentitols by Ring Contraction of Hexopyranose Carbohydrate Derivatives

The excitation of the innermost carbonyl of nono-2,3-diulose derivatives by irradiation with visible-light initiates a sequential Norrish type II photoelimination and aldol cyclization process that finally gives polyfunctionalized cyclopentitols. The rearrangement has been confirmed by the isolation of stable acyclic photoenol intermediates that can be independently cyclized by a thermal 5-(enolexo)-exo-trig uncatalyzed aldol reaction with high diastereoselectivity. In this last step, the large deuterium kinetic isotope effect found for the 1,5-hydrogen atom transfer seems to indicate that the aldol reaction runs through a concerted pericyclic mechanism. Owing to the ready availability of pyranose sugars of various configurations, this protocol has been used to study the influence of pyranose ring-substituents on the diastereoselectivity of the aldol cyclization reaction. In contrast with other pyranose ring contraction methodologies no transition-metal reagents are needed and the sequential rearrangement occurs simply by using visible light and moderate heating (0 to 60 °C).

Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O2. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n−6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n−6 and n−9 fatty acids and by analysis of D-KIE. COX-1 oxidized C20 and C18 fatty acids in the following order of rates: 20:2n−6>20:1n−6>20:3n−9>20:1n−9 and 18:3n−3≥18:2n−6>18:1n−6. 18:2n−6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n−6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n−6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-2H]18:2n−6 with similar D-KIE (~53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n−6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-2H2]- and [11S-2H]18:2n−6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n−6 by COX-1 and 18:1n−9 by 8R-DOX were both accompanied by large D-KIE (>20).

Hydrogen Tunneling Steps in Cyclooxygenase-2 Catalysis

Cyclooxygenases-1 and -2 are tyrosyl radical (Y·)-utilizing hemoproteins responsible for the biosynthesis of lipid-derived autocoids. COX-2, in particular, is a primary mediator of inflammation and believed to be up-regulated in many forms of cancer. Described here are first-of-a-kind studies of COX-2-catalyzed oxidation of the substrate analogue linoleic acid. Very large (≥20) temperature-independent deuterium kinetic isotope effects (KIEs) on the rate constant for enzyme turnover were observed, due to hydrogen atom abstraction from the bisallylic C-H(D) of the fatty acid. The magnitude of the KIE depends on the O(2) concentration, consistent with reversible H/D tunneling mediated by the catalytic Y·. At physiological levels of O(2), retention of the hydrogen initially abstracted by the catalytic tyrosine results in strongly temperature-dependent KIEs on O-H(D) homolysis, also characteristic of nuclear tunneling.

2H Kinetic Isotope Effects and pH Dependence of Catalysis as Mechanistic Probes of Rat Monoamine Oxidase A: Comparisons with the Human Enzyme

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism.

Catalytic Roles of the Metal Ion in the Substrate-Binding Site of Coenzyme B12-Dependent Diol Dehydratase

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K(+) ion; however, QM/MM computations indicate that Ca(2+) ion is more reasonable as the metal ion because calculated Ca-O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K-O distances. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca(2+) ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K(+) ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca(2+) ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C-H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca(2+) ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335.

Pyrolysis of α- and β-Heteroatoms Substituted Ethyl Phenyl Sulfoxides

A study on the mechanism of the thermal decomposition of α- and β-heteroatoms substituted ethyl phenyl sulfoxides was carried out using 1-chloroethyl phenyl sulfoxide (1); two diastereomeric 1-acetoxyethyl (substituted phenyl) sulfoxides (2a) and (2b); and 2-chloroethyl phenyl, 2-bromoethyl phenyl, and 2-methoxyethyl phenyl sulfoxides (3, 4, 5). The rate of pyrolysis of 1 was 4.8 times faster at 160°C than that of ethyl phenyl sulfoxide used as a reference, while those of 2a and 2b were 107 and 155 times faster, respectively. The results indicate that the lone pair of electrons on the α-heteroatoms has a larger rate acceleration effect than the electronegativity of them. The substituent effects of the phenyl group of 2a and 2b gave positive Hammett ρ-values (ρ a = 0.76 and ρ b = 0.80 vs. σ). Activation parameters for 2a and 2b are as follows: 2a, ΔH ‡ = 112 kJmol−1, ΔS ‡ = −20 JK−1mol−1; 2b, ΔH ‡ = 107 kJmol−1, ΔS ‡ = −29 JK-1mol−1. Large deuterium kinetic isotope effects for 1-acetoxyethyl-2,2,2-d3 phenyl sulfoxides (2ad and 2bd) were observed (kH/kD = 3.5 ∼ 4.1). These results suggest that the pyrolysis of α-heteroatom substituted ethyl phenyl sulfoxides proceeds via a five-membered transition state deviated to E1-like in character. On the other hand, from the results of kinetics for the pyrolysis of 3, 4, and 5, no effect by the β-halogen atoms or some deceleration effect by the β-methoxy group was observed. Thus the reaction seems to proceed via an E1-like mechanism.

Cryoreduction of the NO-Adduct of Taurine:α-Ketoglutarate Dioxygenase (TauD) Yields an Elusive {FeNO}8 Species

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent enzymes are a functionally and mechanistically diverse group of mononuclear nonheme-iron enzymes that activate dioxygen to couple the decarboxylation of alphaKG, which yields succinate and CO(2), to the oxidation of an aliphatic C-H bond of their substrates. Their mechanisms have been studied in detail by a combination of kinetic, spectroscopic, and computational methods. Two reaction intermediates have been trapped and characterized for several members of this enzyme family. The first intermediate is the C-H-cleaving Fe(IV)-oxo complex, which exhibits a large deuterium kinetic isotope effect on its decay. The second intermediate is a Fe(II):product complex. Reaction intermediates proposed to occur before the Fe(IV)-oxo intermediate do not accumulate and therefore cannot be characterized experimentally. One of these intermediates is the initial O(2) adduct, which is a {FeO(2)}(8) species in the notation introduced by Enemark and Feltham. Here, we report spectroscopic and computational studies on the stable NO-adduct of taurine:alphaKG dioxygenase (TauD), termed TauD-{FeNO}(7), and its one-electron reduced form, TauD-{FeNO}(8). The latter is isoelectronic with the proposed O(2) adduct and was generated by low-temperature gamma-irradiation of TauD-{FeNO}(7). To our knowledge, TauD-{FeNO}(8) is the first paramagnetic {FeNO}(8) complex. The detailed analysis of experimental and computational results shows that TauD-{FeNO}(8) has a triplet ground state. This has mechanistic implications that are discussed in this Article. Annealing of the triplet {FeNO}(8) species presumably leads to an equally elusive {FeHNO}(8) complex with a quintet ground state.

Aliphatic C−H Bond Activation Initiated by a (μ-η2:η2-Peroxo)dicopper(II) Complex in Comparison with Cumylperoxyl Radical

A (mu-eta(2):eta(2)-peroxo)dicopper(II) complex, [Cu(2)(H-L)(O(2))](2+) (1-O(2)), supported by the dinucleating ligand 1,3-bis[bis(6-methyl-2-pyridylmethyl)aminomethyl]benzene (H-L) is capable of initiating C-H bond activation of a variety of external aliphatic substrates (SH(n)): 10-methyl-9,10-dihydroacridine (AcrH(2)), 1,4-cyclohexadiene (1,4-CHD), 9,10-dihydroanthracene (9,10-DHA), fluorene, tetralin, toluene, and tetrahydrofuran (THF), which have C-H bond dissociation energies (BDEs) ranging from approximately 75 kcal mol(-1) for 1,4-CHD to approximately 92 kcal mol(-1) for THF. Oxidation of SH(n) afforded a variety of oxidation products, such as dehydrogenation products (SH((n-2))), hydroxylated and further-oxidized products (SH((n-1))OH and SH((n-2))=O), dimers formed by coupling between substrates (H((n-1))S-SH((n-1))) and between substrate and H-L (H-L-SH((n-1))). Kinetic studies of the oxidation of the substrates initiated by 1-O(2) in acetone at -70 degrees C revealed that there is a linear correlation between the logarithms of the rate constants for oxidation of the C-H bonds of the substrates and their BDEs, except for THF. The combination of this correlation and the relatively large deuterium kinetic isotope effects (KIEs), k(2)(H)/k(2)(D) (13 for 9,10-DHA, approximately > 29 for toluene, and approximately 34 for THF at -70 degrees C and approximately 9 for AcrH(2) at -94 degrees C) indicates that H-atom transfer (HAT) from SH(n) (SD(n)) is the rate-determining step. Kinetic studies of the oxidation of SH(n) by cumylperoxyl radical showed a correlation similar to that observed for 1-O(2), indicating that the reactivity of 1-O(2) is similar to that of cumylperoxyl radical. Thus, 1-O(2) is capable of initiating a wide range of oxidation reactions, including oxidation of aliphatic C-H bonds having BDEs from approximately 75 to approximately 92 kcal mol(-1), hydroxylation of the m-xylyl linker of H-L, and epoxidation of styrene (Matsumoto, T.; et al. J. Am. Chem. Soc. 2006, 128, 3874).

One-Step versus Stepwise Mechanism in Protonated Amino Acid-Promoted Electron-Transfer Reduction of a Quinone by Electron Donors and Two-Electron Reduction by a Dihydronicotinamide Adenine Dinucleotide Analogue. Interplay between Electron Transfer and Hydrogen Bonding

Semiquinone radical anion of 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ(*-)) forms a strong hydrogen bond with protonated histidine (TolSQ(*-)/His x 2 H(+)), which was successfully detected by electron spin resonance. Strong hydrogen bonding between TolSQ(*-) and His x 2 H(+) results in acceleration of electron transfer (ET) from ferrocenes [R2Fc, R = C5H5, C5H4(n-Bu), C5H4Me] to TolSQ, when the one-electron reduction potential of TolSQ is largely shifted to the positive direction in the presence of His x 2 H(+). The rates of His x 2 H(+)-promoted ET from R2Fc to TolSQ exhibit deuterium kinetic isotope effects due to partial dissociation of the N-H bond in His x 2 H(+) at the transition state, when His x 2 H(+) is replaced by the deuterated compound (His x 2 D(+)-d6). The observed deuterium kinetic isotope effect (kH/kD) decreases continuously with increasing the driving force of ET to approach kH/kD = 1.0. On the other hand, His x 2 H(+) also promotes a hydride reduction of TolSQ by an NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2). The hydride reduction proceeds via the one-step hydride-transfer pathway. In such a case, a large deuterium kinetic isotope effect is observed in the rate of the hydride transfer, when AcrH2 is replaced by the dideuterated compound (AcrD2). In sharp contrast to this, no deuterium kinetic isotope effect is observed, when His x 2 H(+) is replaced by His x 2 D(+)-d6. On the other hand, direct protonation of TolSQ and 9,10-phenanthrenequinone (PQ) also results in efficient reductions of TolSQH(+) and PQH(+) by AcrH2, respectively. In this case, however, the hydride-transfer reactions occur via the ET pathway, that is, ET from AcrH2 to TolSQH(+) and PQH(+) occurs in preference to direct hydride transfer from AcrH2 to TolSQH(+) and PQH(+), respectively. The AcrH2(*+) produced by the ET oxidation of AcrH2 by TolSQH(+) and PQH(+) was directly detected by using a stopped-flow technique.

Real-Time Monitoring of the Oxalate Decarboxylase Reaction and Probing Hydron Exchange in the Product, Formate, Using Fourier Transform Infrared Spectroscopy

Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [(13)C]oxalate as the substrate. The K(m) for oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidase activity by the lack of formate being produced by the latter. In D(2)O, the product with oxalate decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on V(max) (7.1 +/- 0.3), K(m) for oxalate (3.9 +/- 0.9), and k(cat)/K(m) (1.8 +/- 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.

Kinetic and Chemical Mechanisms of the fabG-Encoded Streptococcus pneumoniae β-Ketoacyl-ACP Reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.