Hydrogen Transfer Step Research Articles

On the Parallel Mechanism of the Dissociation of Energy-Selected P(CH3)3+ Ions

Energy selected trimethyl phosphine ions were prepared by threshold photoelectron photoion coincidence (TPEPICO) spectroscopy. This ion dissociates via H, CH(3), and CH(4) loss, the latter two involving hydrogen transfer steps. The ion time-of-flight distribution and the breakdown diagram are analyzed in terms of the statistical RRKM theory, which includes tunneling. Ab initio and DFT calculations provide the vibrational frequencies required for the RRKM modeling. CH(3) loss could produce both the P(CH(3))(2)(+) by a simple bond dissociation step, and the more stable HP(CH(2))CH(3)(+) ion by a hydrogen transfer step. Quantum chemical calculations are extensively used to uncover the reaction scheme, and they strongly suggest that the latter product is exclusively formed via an isomerization step in the energy range of the experiment. The data analysis, which includes modeling with the trimethyl phosphine thermal energy distribution, provides accurate onset energies for both H (E(0K) = 1024.1 +/- 3.5 kJ/mol) and CH(3) (E(0K) = 1024.8 +/- 3.5 kJ/mol) loss reactions. From this analysis, we conclude that the Delta(f)H(298K) degrees [HP(CH(2))(CH(3))(+)] = 783 +/- 8 kJ/mol and Delta(f)H(298K) degrees [P(CH(2))(CH(3))(2)(+)] = 711 +/- 8 kJ/mol.

The effect of catalyst properties on the in situ reduction of sulfur in FCC gasoline

In this paper, the results of a study on the effects of catalyst properties and contaminant metals on FCC gasoline sulfur are reported. Data for four steamed deactivated FCC catalysts showed that the higher the unit cell size (UCS) of an FCC catalyst, the lower the sulfur concentration in FCC gasoline. These results suggest a mechanism that explains gasoline sulfur reduction through a hydrogen transfer step resulting in saturation of thiophenes. The effects of contaminant metals, that usually exist on FCC catalysts (Ni and V), on FCC gasoline sulfur, were examined for both equilibrium and deactivated catalysts. The presence of these metals lowers gasoline sulfur dramatically. Vanadium (V) in its highest oxidation step is most effective. It appears that these metals catalyze the cyclization of long alkyl-thiophenes to form alkyl-benzothiophenes boiling outside the gasoline range. Finally, it was observed that the presence of ZSM-5 additive resulted in an increase in gasoline sulfur.

Density functional theory studies of mechanistic aspects of the SCR reaction on vanadium oxide catalysts

Density functional theory (DFT) calculations were carried out on a vanadium oxide cluster containing four vanadium atoms to probe the mechanism of the selective catalytic reduction (SCR) of NO with ammonia. The interaction of ammonia with Brønsted acid sites on this V 4-cluster leads to the formation of NH 4 species bonded to two vanadyl (VO) groups, with a bonding energy of −110 kJ/mol. This adsorbed NH 4 species reacts with NO in a series of steps to form an adsorbed NH 2NO species, which subsequently undergoes decomposition to form N 2, H 2O, and a reduced vanadium oxide cluster (V 4H). The latter reaction occurs via a series of hydrogen-transfer steps by a “push–pull” mechanism with adjacent VO and VOH groups on the vanadium oxide cluster. The rate limiting process in this conversion of NO and NH 3 to give N 2, H 2O, and V 4H involves the reaction of an adsorbed NH 3NHO adduct to form NH 2NO species. The transition state of this step may be stabilized through hydrogen bonding with surrounding vanadia and/or titania moieties.

Theoretical Studies of the Cross-Linking Mechanisms between Cytosine and Tyrosine

DNA-protein cross-linking is one of the many DNA lesions mediated by hydroxyl radicals, the most damaging among the reactive oxygen species in biological systems. Density functional theory methods are employed to investigate the complex reaction mechanisms of the formation of cytosine-tyrosine cross-links as observed in gamma-irradiated aqueous solutions of cytosine and tyrosine, as well as in gamma-irradiated nucleohistone. The majority of the radical addition mechanisms considered are found to have significant barriers and therefore to be thermodynamically unfavorable for the formation of the initial cross-linked product. Our calculated reaction potential energy surfaces suggest that a feasible complete mechanism consists of radical combination forming the initial cross-linked product, a hydrogen shuffle within the initial cross-linked product, and an acid-catalyzed dehydration reaction. Water and hydrogen-bonding interactions are suggested to play a key role in catalyzing the hydrogen-transfer step of the reaction.

Thermal reduction of 7H-benz[d,e]anthracen-7-one and related ketones under hydrogen-transfer conditions.

In the presence of hydrogen donor solvents and at elevated temperatures, aromatic ketones can be selectively deoxygenated to the corresponding hydroaromatic compounds. The kinetics for reduction of 7H-benz[d,e]anthracen-7-one (benzanthrone, 6) into 7H-benz[d,e]anthracene (benzanthrene, 1) in 9,10-dihydroanthracene (3) solvent has been investigated in detail. The relatively slow hydrogenation of 6 is due to reversibility of the initial hydrogen-transfer step according to a reverse radical disproportionation (RRD). The dynamics could well be rationalized using the energetics of species computed by density functional theory (DFT). The application of hydrogen donors such as 1 as a hydrogen-transfer agent, although favorable in terms of a low benzylic carbon-hydrogen bond dissociation enthalpy, is limited due to the slow self-hydrogenation, which in case of 1 gives 5,6-dihydro-4H-benz[d,e]anthracene (7).

Synthesis of propionate motifs: diastereoselective tandem reactions involving anionic and free radical based processes.

Reported herein is a strategy employing a Mukaiyama reaction in tandem with a hydrogen transfer reaction for the elaboration of propionate motifs. The nature of the protecting groups on the chiral beta-alkoxy aldehyde and the type of Lewis acid used are varied to modulate the stereochemical outcome of the tandem reactions. The mode of complexation is thus controlled (monodentate or chelate) for the Mukaiyama reaction to give access to either syn or anti aldol products, precursors of the free radical reduction reaction. The endocyclic effect is subsequently capitalized upon to control the hydrogen transfer step so that the syn-reduced product may be achieved. Proceeding with excellent yield and diastereoselectivity, the synthetic sequence proposed gives access to syn-syn and syn-anti propionate motifs. Also considered is a complementary approach using a chelation-controlled Mukaiyama reaction in tandem with a free radical allylation reaction under the control of the endocyclic effect that leads to the anti-anti product.

A Langmuir–Hinshelwood model for a hydrogen transfer mechanism in the selective hydrogenation of acetylene over a Pd/γ-Al 2O 3 catalyst prepared by the sol–gel method

This study represents an effort to fit a Langmuir–Hinshelwood-type mechanism which involves a proposed hydrogen transfer step from a carbonaceous deposit with observed rate data, spectroscopic data, and reported literature results on a supported Pd/γ-Al 2O 3 catalyst prepared by the sol–gel method. Our data was taken in a differential reactor as the temperature was varied from 100 to 225°C in six increments. A change in the apparent reactant orders and activation energies support a multiple mechanism rate expression. Our data correlates best with an expression composed of two mechanisms: a competitive, low-temperature mechanism (mechanism I) with an activation energy of 29 kJ/mol, and a non-competitive, high-temperature mechanism (mechanism II) with an activation energy of 168 kJ/mol. Both mechanisms were fit using an enthalpy of adsorption of −124 kJ/mol for acetylene. The enthalpy of the hydrogen transfer was found to be 133 kJ/mol, indicating an endothermic process. Mechanism II is predicted to emerge and dominate at high temperatures.

Hydrogen transfer in hydrocarbon conversions over transition metal surfaces

Hydrogen-transfer steps are a poorly studied and rarely proposed class of elementary reactions in heterogeneous catalysis over metal surfaces. In these steps, a hydrogen atom that is attached to the carbon in one surface species transfers to a carbon atom of another species. Unity bond index–quadratic exponential potential (UBI–QEP) calculations of the activation energies for three model surfaces representing substrates with strong, weak, and intermediate chemisorption abilities, combined with UBI–QEP/DFT data for the Pt(1 1 1) surface, suggest that there is no great energetic impediment to these steps. Therefore, they should not be arbitrarily ignored when building mechanisms of hydrocarbon conversions over metal surfaces. Moreover, kinetics simulations show that conditions are possible under which one-step hydrogen transfer is faster than the two-step process with the formation of adsorbed hydrogen.

Organic chemistry of coke formation

The modes of formation of carbonaceous deposits (“coke”) during the transformation of organic compounds over acid and over bifunctional noble metal-acid catalysts are described. At low reaction temperatures, (<200°C) “coke” formation involves mainly condensation and rearrangement steps. Therefore, the deposits are not polyaromatic and their composition depends very much on the reactant. The retention of the “coke” molecules on the catalysts is mainly due to their strong adsorption and to their low volatility (gas-phase reactions) or to their low solubility (liquid-phase reactions). At high temperatures (>350°C), the coke components are polyaromatic. Their formation involves hydrogen transfer (acid catalysts) and dehydrogenation (bifunctional catalysts) steps in addition to condensation and rearrangement steps. On microporous catalysts, the retention of coke molecules is due to their steric blockage within the micropores.

The Coenzyme B12 Analog 5′-Deoxyadenosylcobinamide-GDP Supports Catalysis by Methylmalonyl-CoA Mutase in the Absence of Trans-ligand Coordination

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 Å and 2.1 Å resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296–301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114–121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of ω-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 Å resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.

A Theoretical Study of the Molecular Mechanism for the Carboxylation Chemistry in Rubisco

Stereochemical and structural aspects for the carbon dioxide fixation followed by hydration of d-ribulose 1,5-bisphosphate catalyzed by Rubisco are analyzed by using two model substrates, hydroxypropanone and 3,4-dihydroxy-2-pentanone. The molecular mechanisms for the carboxylation, C3-hydration and C2-inversion processes, are theoretically characterized and discussed with saddle points of index 1 representing transition structures (TS) and relevant (molded) intermediates mirroring the experimentally proposed mechanism. Ab initio SCF MO calculations at 3-21G and 6-31G** basis set levels of theory were used, while the correlation energy is included at the MP2/6-31G** level. The mapping starts from TS1, the carboxylation transition structure, which describes now the coupling of the carbon dioxide attack to the substrate C2-center in its dienol form with a synchronous interconversion of the C3 hydroxyl into a ketone group. Thereafter, water addition leads to a gem-diol followed by another step of intramolecular hydrogen transfer coupled with the C2−C3 bond breaking process. This TS breaks into one model of 3-d-phosphoglycerate product and an intermediate. The configuration inversion at the C2-center is found to be possible via intramolecular hydrogen transfer, as suggested by the corresponding TS relating the intermediate to the C2-inverted conformation. The complete set of steps found gives a self-contained description of the carboxylation followed by hydrolysis with proper stereochemistry. Comparisons between the transition state analogue: 2-carboxy-d-arabinitol-1,5-bisphosphate and TS1 show that both structures can be superposed with minimal root-mean-square deviation for C-atoms. All other calculated stationary TSs share the gross conformational features of TS1, and consequently, all molecular rearrangements detected in a vacuum can be accommodated without constraints into the active site of Rubisco. Transition structures invariances with respect to level of theory and molecular models are discussed.

Selective transformation of methanol into light olefins over a mordenite catalyst: reaction scheme and mechanism

The conversion of a dimethylether–water mixture into hydrocarbons was investigated at 530°C over a commercial mordenite catalyst. A dimethylether–water mixture was chosen instead of methanol in order to limit thermal effects due to the exothermicity of the transformations of methanol into dimethylether and into hydrocarbons. The main reaction products are methanol, C 1–C 12 hydrocarbons and coke, which is responsible for catalyst deactivation. The distribution of the products was determined on the fresh catalyst over a large range of conversions (up to 100%), which allowed us to propose a complete reaction scheme. The conversion of dimethylether into hydrocarbons is faster than that of methanol, which is formed rapidly by the hydration of dimethylether. Whereas C 3–C 7 alkenes appear as primary products, ethylene results from secondary cracking. Propene would be formed from a Stevens rearrangement of oxonium ions, and higher alkenes from the rapid methylation of propene. C 6–C 7 alkenes and probably higher alkenes appearing in traces are transformed into aromatics through cyclization and hydrogen transfer steps, with the simultaneous formation of C 3–C 4 alkenes. The resulting aromatics are rapidly alkylated by dimethylether or methanol. Methane is formed directly from dimethylether or methanol, but also from the demethylation of polymethylbenzenes or of coke molecules.

Lanthanide (Eu or Yb)-promoted Ni, Cu and Ag-catalyzed transfer hydrogenation with NH 3 as a hydrogen donor

Novel lanthanide (Ln)-promoted Ni, Cu and Ag catalysts prepared by the reaction of Ni/SiO 2, Cu/SiO 2 and Ag/ZrO 2 with the dissolved Eu or Yb metals in liquid ammonia have been studied for the catalytic transfer hydrogenation of ethene, buta-1,3-diene and propyne. The transfer hydrogenation with the aid of ammonia as a hydrogen donor was greatly enhanced by the Ln-promoted catalysts. The catalytic activity for the transfer hydrogenation increased with increasing lanthanide content in the bimetallic catalysts, which evidently reflected synergetic effects between the lanthanide and transition metals. The transfer hydrogenation of ethene with ammonia over Eu–Ni/SiO 2 proceeded to form ethane and nitrogen, maintaining the following stoichiometric relationships: 3C 2H 4+2NH 3→3C 2H 6+N 2 The catalytic hydrogen transfer from ammonia to buta-1,3-diene or propyne similarly followed the stoichiometric relationships. The Ln-promoted catalysts showed substrate selectivity for the transfer hydrogenation using ammonia as the hydrogen source; Ln–Ni/SiO 2 was reactive for ethene and buta-1,3-diene, but not for propyne. Ln–Cu/SiO 2 was active for propyne and buta-1,3-diene. Ln–Ag/SiO 2 was relatively active for buta-1,3-diene. The major factors governing the catalytic transfer hydrogenation seem to be involved in the participation of the acceptor and donor molecules in the hydrogen transfer step rather than the ease of dehydrogenation of ammonia and the processes of hydrogen addition to the acceptor. The catalytic hydrogen transfer from ammonia to acceptor molecules occurs concertedly.

Structural and Catalytic Characterization of Solid Acids Based on Zirconia Modified by Tungsten Oxide

Tungsten oxide species form strong acid sites on ZrO2supports and inhibit ZrO2crystallite sintering and tetragonal to monoclinic structural transformations. W-LIX-ray absorption near-edge spectra suggest that the W centers are in a distorted octahedral oxygen environment, even after dehydration at 673 K, in all WOx–ZrO2samples (2–21 wt.% W) oxidized at 1073 K. Maximumo-xylene isomerization turnover rates (per W atom) on WOx–ZrO2solids occur at WOxsurface densities (10 W nm−2) that exceed the theoretical monolayer capacity of ZrO2. Similar turnover rates are obtained on WOx–ZrO2samples with similar WOxsurface densities (W nm−2) over a large range of oxidation temperatures (773–1223 K) and WOxconcentrations (5–21 wt.% W). UV–visible spectra suggest an increase in WOxdomain size with increasing surface density. High isomerization turnover rates appear to require the presence of WOxdomains of intermediate size on ZrO2surfaces. WOxdomains of intermediate size appear to provide a compromise between reducibility and accessibility of WOxcenters. These domains are necessary to delocalize a temporary charge imbalance that forms Brønsted acid sites in the presence of H2and stabilizes carbocation intermediates. The presence of H2duringo-xylene isomerization increases turnover rates and prevents rapid deactivation. Slow D2/o-xylene exchange reactions indicate that H atoms from H2are not frequently involved in the activation or desorption of xylenes. H2is required, however, in order to reverse the occasional desorption of H atoms duringo-xylene isomerization reactions. These desorption processes lead to the destruction of Brønsted acid sites by the formation of strongly adsorbed unsaturated species in the absence of H2. After promotion with Pt (0.3 wt.%), WOx–ZrO2solids catalyzen-heptane isomerization in the presence of H2at 400–500 K with much higher selectivity than sulfated oxides or zeolitic acids at similar turnover rates. On Pt/WOx–ZrO2, efficient hydrogen transfer steps prevent extensive cracking of adsorbed carbocations by limiting their surface lifetimes.

Tertiary recycling of polyethylene: mechanism of liquid production from polyethylene by thermolysis/reactive distillation

Thermolysis coupled with reactive distillation was studied as a potential process for the tertiary recycling of polyethylene. The reactions were carried out at 440°C and under a nitrogen atmosphere. Processing under these conditions allowed for a higher conversion of polyethylene into a condensable liquid product than is usually reported with conventional pyrolysis. The products obtained from this study were characterized by a high concentration of straight-chain α-olefins. The relative importance of the various steps in the mechanism of thermal degradation of polyethylene was found to be very different from that observed in other studies employing just thermolysis or pyrolysis. In addition to molecular weight reduction by random scission, intermolecular and intramolecular hydrogen transfer steps were found to be very important to the production of liquid. Intramolecular hydrogen transfer followed by β-scission was also found to be an important mechanism for the production of short chain length α-olefins.

Radical Chemistry under Diffusional Constraints: Impact of Spacer Molecules on the Thermolysis of Surface-Immobilized Bibenzyl

Silica nanoparticle surfaces have been chemically modified to contain a molecular probe, 1,2-diphenylethane (bibenzyl), as well as a second spacer molecule of variable structure. Thermolysis kinetics and products at 400 °C have been determined for bibenzyl, and the impact of diffusional constraints and spacer molecular structure on the multipathway radical chemistry has been analyzed relative to fluid phases. Unimolecular homolysis rate constants, k = (7−9) × 10-6 s-1, are found to be independent of spacer molecular structure (naphthalene, diphenylmethane, tetralin) and similar to values measured in fluid phases, indicating the lack of a cage effect on the silica surface. However, the total rate of bibenzyl thermolysis and the resulting product selectivities are profoundly affected by both surface immobilization and the structure of the spacer molecule. Naphthalene spacers behave as “molecular walls” serving as physical barriers to bimolecular hydrogen transfer steps on the surface and augmenting the effects of diffusional constraints. In contrast, diphenylmethane spacers are found to serve as hydrogen transfer, radical relay catalysts that translocate radical sites across the surface and diminish the impact of diffusional constraints. Tetralin spacers undergo significant reaction with free-radical intermediates, selectively producing the isomeric methylindan via a chain pathway that is promoted by the restrictions on diffusion.

Skeletal Isomerization ofn-Butenes: II. Composition, Mode of Formation, and Influence of Coke Deposits on the Reaction Mechanism

On a fresh HFER zeolite (Si/Al=13.8) at 623 K, the skeletal isomerization ofn-butenes is accompanied by various reactions: disproportionation into propene and pentenes, coking, hydrogen transfer (in the decreasing order of significance). While there is a rapid deactivation of disproportionation, coking, and hydrogen transfer reactions, owing to the blockage of the pores by carbonaceous compounds (coke), an increase in the rate of isomerization is at first observed followed by a decrease at long time-on-stream. At the maximum of isomerization only 10% of the pore volume remains accessible to nitrogen adsorbent. Moreover, IR spectroscopy shows that 75% of the OH groups are in interaction with coke molecules, the remaining 25% being inaccessible to ammonia and, hence, to the reactant. Coke was found to be composed of slightly condensed aromatics (with two to four aromatic rings) trapped at the intersections of the eight- and 10-membered-ring channels. Coke molecules result from the transformation of secondary benzenic products, slowly desorbed from the zeolite pores, through two different ways, (i) the classical route via alkylation, cyclization, and hydrogen transfer steps and (ii) dehydrogenative coupling. The large significance of this latter reaction in the formation of HFER coke molecules in comparison to the other zeolites, can be related to the proximity of adjacent channel intersections. To explain the initial increase in the rate of butene isomerization a new reaction process is proposed involving as active sites benzylic carbocations formed from coke molecules trapped in pores located near the outer surface of the crystallites (pore mouth catalysis). The deactivation of this reaction which occurs at long time-on-stream is shown to be due to the growth of the active coke molecules.

Kinetics and Thermodynamics of Hydrogen Atom Transfer to Superoxometal Complexes

Dicationic metal superoxo complexes, L(H2O)MOO2+ (M = Rh, Cr; L = (NH3)4, (H2O)5, [14]aneN4) react with rhodium hydrides L‘(H2O)RhH2+ (L‘ = (NH3)4, [14]aneN4, and meso-Me6-[14]aneN4) to yield L(H2O)MOOH2+ and L‘(H2O)2Rh2+. The rhodium(II) product is rapidly converted to L‘(H2O)RhOO2+ in a reaction with molecular oxygen (k ∼ 108 M-1 s-1), or to a binuclear superoxo product in the absence of O2. When the central metal atom and nonparticipating ligands are identical in the two reactants (M = Rh, L = L‘), the O2 insertion into Rh−H bond becomes catalytic, L‘RhH2+ + O2 → L‘RhOOH2+ (L‘RhOO2+ = catalyst). The hydrogen transfer step exhibits a large kinetic isotope effect (kH/kD = 7.6 for the reaction of CraqOO2+ with ([14]aneN4)(H2O)RhH2+, ΔHH⧧ = 20.3 ± 1.8 kJ mol-1, ΔSH⧧ = −136 ± 6 J mol-1 K-1, and ΔHD⧧ = 24.7 ± 4.3 kJ mol-1, ΔSD⧧ = −138 ± 15 J mol-1 K-1), but the isotopic composition of the solvent has no effect on rates, kH2O/kD2O = 1. Steric effects play a major role in the kinetics. From the available therm...

Involvement of Glu-264 and Arg-235 in the essential interaction between the catalytic imidazole and substrate for the D-lactate dehydrogenase catalysis.

For Lactobacillus pentosus D-lactate dehydrogenase, the binding of 2-ketoacids is markedly stabilized through interactions between the protonated imidazole of His-296, an acid/base catalyst of the enzyme, and the carbonyl oxygen of 2-ketoacids. The replacement of Arg-235 with Gln destabilized the inhibitory binding of oxamate much more than that of formate, acetate, or propionate, and the Arg to Lys substitution specifically diminished only oxamate binding. On the other hand, replacement of a conserved Glu, Glu-264, with Gln severely impaired the enzyme activity and markedly reduced affinity to 2-keto acids. The pH dependence of the oxamate inhibition revealed that the substitutions of Arg-235 and Glu-264 induced a great loss of the imidazole-carbonyl interaction. However, replacement of Glu-264 with Asp, another acidic amino acid, affected the enzyme function less than the Glu to Gln substitution. In addition, both the Arg-235 and Glu-264 substitutions induced marked increases in the primary isotope effect on the catalysis, suggesting that these amino acids stimulate the hydrogen transfer step in the catalysis. We concluded, therefore, that the guanidino and carboxyl groups of Arg-235 and Glu-264, respectively, cooperatively promote the essential imidazole-substrate interaction, enhancing the substrate binding and catalysis.