Rate Of Hydride Transfer Research Articles

Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor-acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD's rearrangement toward tunneling-ready states. This structure-function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C-H→C transfer reactions.

Acid strength and solvation effects on methylation, hydride transfer, and isomerization rates during catalytic homologation of C1 species

Dimethyl ether (DME) homologation forms isobutane and triptane (2,2,3-trimethylbutane) with supra-equilibrium selectivities within C4 and C7 hydrocarbons on both mesoporous solid acids (SiO2–Al2O3, H3PW12O40/SiO2) and the acid forms of various zeolites (BEA, FAU, MFI) via methylation and hydride transfer steps that favor isobutane and triptane formation because of the relative stabilities of ion-pairs at transition states for chains along the preferred growth path. The stabilities of ion-pair transition states increase as acid sites become stronger and energies for charge separation decrease and as van der Waals interactions within pores become stronger, which respectively lead to higher rates on H3PW12O40/SiO2 and aluminosilicate zeolites than on amorphous SiO2–Al2O3. Solid acids with different strengths and abilities to solvate ion-pairs by confinement differ in selectivity because strength and solvation influence transition states for the hydride transfer, methylation, and isomerization steps to different extents. Stronger acid sites on H3PW12O40/SiO2 favor isomerization and hydride transfer over methylation; they lead to higher selectivities to n-butane and non-triptane C7 isomers than the weaker acid sites on BEA, FAU, and mesoporous SiO2–Al2O3. This preference for hydride transfer and isomerization on stronger acids reflects transition states with more diffuse cationic charge, which interact less effectively with conjugate anions than more localized cations at methylation transition states. The latter recover a larger fraction of the energy required to form the ion-pair, and their stabilities are less sensitive to acid strength than for diffuse cations. Large-pore zeolites (BEA, FAU) form triptane with higher selectivity than SiO2–Al2O3 because confinement within large pores preferentially solvates the larger transition states for hydride transfer and methylation, which preserve the four-carbon backbone in triptane, over smaller transition states for alkoxide isomerization steps, which disrupt this backbone and cause growth beyond C7 chains and subsequent facile β-scission to form isobutane. MFI forms isobutane and triptane with much lower selectivity than mesoporous acids or large-pore zeolites, because smaller pores restrict the formation of bimolecular methylation and hydride transfer transition states required for chain growth and termination steps to a greater extent than those for monomolecular alkoxide isomerization. These data and their mechanistic interpretations show that the selective formation of isobutane and triptane from C1 precursors like DME is favored on all acids as a result of the relative stability of methylation, hydride transfer, and isomerization transition states, but to a lesser extent when small confining voids and stronger acid sites preferentially stabilize monomolecular isomerization transition states. The observed effects of acid strength and confinement on rates and selectivities reflect the more effective stabilization of all ion-pairs on stronger acids and within solvating environments, but a preference for transition states with more diffuse charge on stronger acids and for ion-pairs with the appropriate solvation within voids of molecular dimensions.

Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 (“stomach” isoenzyme) are substantially larger (10–100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD + and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD + and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

The Cys319 Loop Modulates the Transition between Dehydrogenase and Hydrolase Conformations in Inosine 5′-Monophosphate Dehydrogenase

X-ray crystal structures of enzyme-ligand complexes are widely believed to mimic states in the catalytic cycle, but this presumption has seldom been carefully scrutinized. In the case of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase (IMPDH), 10 structures of various enzyme-substrate-inhibitor complexes have been determined. The Cys319 loop is found in at least three different conformations, suggesting that its conformation changes as the catalytic cycle progresses from the dehydrogenase step to the hydrolase reaction. Alternatively, only one conformation of the Cys319 loop may be catalytically relevant while the others are off-pathway. Here we differentiate between these two hypotheses by analyzing the effects of Ala substitutions at three residues of the Cys319 loop, Arg322, Glu323, and Gln324. These mutations have minimal effects on the value of k(cat) (≤5-fold) that obscure large effects (>10-fold) on the microscopic rate constants for individual steps. These substitutions increase the equilibrium constant for the dehydrogenase step but decrease the equilibrium between open and closed conformations of a mobile flap. More dramatic effects are observed when Arg322 is substituted with Glu, which decreases the rates of hydride transfer and hydrolysis by factors of 2000 and 130, respectively. These experiments suggest that the Cys319 loop does indeed have different conformations during the dehydrogenase and hydrolase reactions as suggested by the crystal structures. Importantly, these experiments reveal that the structure of the Cys319 loop modulates the closure of the mobile flap. This conformational change converts the enzyme from a dehydrogenase into hydrolase, suggesting that the conformation of the Cys319 loop may gate the catalytic cycle.

ChemInform Abstract: Kinetics of Hydride Transfer Reactions from Hydrosilanes to Carbenium Ions. Substituent Effects in Silicenium Ions

Rates of hydride transfer from hydrosilanes HSiR 1 R 2 R 3 with widely varying substitution to para-substituted diarylcarbenium ions have been measured in dichloromethane solution. Generally the reactions follow a second-order rate law, -d[Ar 2 CH + ]/dt=k 2 [Ar 2 CH + ] [HSiR 1 R 2 R 3 ], and k 2 is independent of the degree of ion-pairing and the nature of the counterion (exceptions are reported). The reaction rates are almost independent of solvent polarity

Ligand-induced changes in the conformational stability and flexibility of glutamate dehydrogenase and their role in catalysis and regulation

Bovine glutamate dehydrogenase (GDH) is allosterically regulated and requires substrate-induced subunit interactions for maximum catalytic activity. Steady-state and presteady-state kinetics indicate that the rate-limiting step depends on the nature of the substrate and are likely associated with conformational fluctuations necessary for optimal hydride transfer. Deuterated glutamate shows a steady-state isotope effect but no effect on the presteady-state burst rate, demonstrating that conformational effects are rate limiting for hydride transfer while product release is overall rate limiting for glutamate. Guanidine hydrochloride unfolding, heat inactivation, and differential scanning calorimetry demonstrate the effects of alternative substrates, glutamate and norvaline, on conformational stability. Glutamate has little effect on overall stability, whereas norvaline markedly stabilizes the protein. Limited proteolysis demonstrates that glutamate had a variety of effects on local flexibility, whereas norvaline significantly decreased conformational fluctuations that allow protease cleavage. Dynamic light scattering suggests that norvaline stabilizes all interfaces in the hexamer, whereas glutamate had little effect on trimer-trimer interactions. The substrate glutamate exhibits negative cooperativity and complex allosteric regulation but has only minor effects on global GDH stability, while promoting certain local conformational fluctuations. In contrast, the substrate norvaline does not show negative cooperativity or allow allosteric regulation. Instead, norvaline significantly stabilizes the enzyme and markedly slows or prevents local conformational fluctuations that are likely to be important for cooperative effects and to determine the overall rate of hydride transfer. This suggests that homotropic allosteric regulation by the enzymatic substrate involves changes in both global stability and local flexibility of the protein.

Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP +-reductase

Two transient charge–transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP + reductase (FNR) with NADP +/H, FNR ox-NADPH (CTC-1), and FNR rd-NADP + (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP +/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O(2) of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H(2)O(2). Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O.sugar complex. Transient kinetics of wild-type (WT) and Thr(169) --> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K(d) (45-47 mm), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k(red). In the crystal structure of T169S, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr(169) side chain are required for intermediate stabilization.

The Effect of Osmolytes on Protein Dynamics in LDH-Catalyzed Reaction

Laser induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the dynamics of LDH-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the LDH reaction system in presence of varying amounts of trimethylamine-N-oxide (TMAO), a protein stabilizing osmolyte, or urea, a protein destabilizing osmolyte. The fluorescence kinetics show that TMAO does not significantly affect pyruvate binding/unbinding, but noticeably changes the dynamics of the active site loop, strongly decreasing its kinetic response. The nature of these changes is not yet fully understood. The lower rate of the absorption kinetics, representing mainly the hydride transfer step and to a much lesser extent the loop dynamics, becomes faster upon TMAO addition, especially at lower temperatures (17-22C). This may be explained by an enhancement of hydrogen tunneling resulting from compression of the active site. Urea, in concentrations up to 1 M, strongly diminishes the kinetic response of pyruvate binding/unbinding, while increasing its rate at higher temperatures (32-37C). Loop motions slow in presence of urea, as does the rate of hydride transfer. In presence of 2 M urea, no kinetic features arising from the reaction are observed, presumably due to expected protein denaturation.

Evidence To Support the Hypothesis That Promoting Vibrations Enhance the Rate of an Enzyme Catalyzed H-Tunneling Reaction

In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.

Plasmodium falciparum Ferredoxin-NADP+ Reductase His286 Plays a Dual Role in NADP(H) Binding and Catalysis

The NADP-binding site of Plasmodium falciparum ferredoxin-NADP(+) reductase contains two basic residues, His286 and Lys249, conserved within the Plasmodium genus, but not in other plant-type homologues. Previous crystal studies indicated that His286 interacts with the adenine ring and with the 5'-phosphate of 2'-P-AMP, a ligand that mimics the adenylate moiety of NADP(H). Here we show that replacement of His286 with aliphatic residues results both in a decrease in the affinity of the enzyme for NADPH and in a decrease in k(cat), due to a lowered hydride-transfer rate. Unexpectedly, the mutation to Gln produces an enzyme more active than the wild-type one, whereas the change to Lys destabilizes the nicotinamide-isoalloxazine interaction, decreasing k(cat). On the basis of the crystal structure of selected mutants complexed with 2'-P-AMP, we conclude that the His286 side chain plays a dual role in catalysis both by providing binding energy for NADPH and by favoring the catalytically competent orientation of its nicotinamide ring. For the latter function, the H-bonding potential rather than the positively charged state of the His286 imidazole seems sufficient. Furthermore, we show that the Lys249Ala mutation decreases K(m)(NADPH) and K(d) for NADP(+) or 2'-P-AMP by a factor of 10. We propose that the Lys249 side chain participates in substrate recognition by interacting with the 2'-phosphate of NADP(H) and that this interaction was not observed in the crystal form of the enzyme-2'-P-AMP complex due to a conformational perturbation of the substrate-binding loop induced by dimerization.

Modeling of key reaction pathways: Zeolite catalyzed alkylation processes

A six lump kinetic model that considers the key reactions for the zeolite catalyzed alkylation process is presented. The influence of different reactions and rate limiting steps on reactor performance is examined by coupling an appropriate reactor model that accounts for different back-mixing on reactor scale, with a zeolite particle model which accounts for the diffusion inside the zeolite pore, the alkylation reaction, and zeolite deactivation. Model predictions are compared with experimental results and lead to conclusions that hydride transfer and oligomerization reactions are the key kinetic steps affecting the overall performance of zeolite catalyzed alkylation processes. It is suggested that higher alkylate yield and longer zeolite activity are achieved by increasing the intrinsic hydride transfer rate and the ratio of feed isobutane to n-butene (P/O) concentration. For a given P/O feed ratio, achieving close to plug flow for isobutane and high back-mixing for n-butene further enhances local P/O ratio and yield. Furthermore, optimal zeolite catalyst design should consider the egg shell type of Brønsted acid site distribution and a lower silicon to alumina (Si/Al) ratio.

Effect of Dimerization on the Stability and Catalytic Activity of Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima

In contrast to all other chromosomally encoded dihydrofolate reductases characterized so far, dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima forms a highly stable dimer. The dimer interface involves residues whose mobility is important for catalysis in monomeric DHFRs. Here, we report the generation of a variant of DHFR from T. maritima, TmDHFR-V11D, in which a single amino acid replacement was sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric TmDHFR-V11D was 15 kJ mol(-1) lower than that of the wild-type dimer, while the melting temperature of monomeric TmDHFR-V11D was comparable to that of monomeric DHFR from the thermophile Bacillus stearothermophilus, supporting the hypothesis that oligomerization is required to achieve the thermal stabilities necessary for activity at temperatures optimal for growth of hyperthermophiles. Both the steady-state turnover numbers and rates of hydride transfer were reduced in TmDHFR-V11D. However, a similar reduction of the rate constants was observed in a different variant, TmDHFR-V126E, which remained as a dimer under all experimental conditions used here. Monomeric TmDHFR-V11D had a similar rate of hydride transfer to the dimeric form, but a reduced steady-state turnover rate. Intersubunit motions therefore appear to be less important than correlated motions within individual subunits for TmDHFR-catalyzed hydride transfer, but are critical to the overall progression of the catalytic cycle. Hence, the reduced catalytic activity of TmDHFR relative to the monomeric Escherichia coli enzyme is not caused by rigidity resulting from dimerization, but is a subtle consequence of the sequence and structure of its subunits, which appear to have evolved to allow thermostability at the expense of catalysis.

Parallel Pathways and Free‐Energy Landscapes for Enzymatic Hydride Transfer Probed by Hydrostatic Pressure

Mutation of an active-site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor-acceptor distance in the wild-type enzyme, increased conformational freedom "dampens" its effect in the mutant.We show that hydride transfer from NADH to FMN catalysed by the N189A mutant of morphinone reductase occurs along parallel "chemical" pathways in a conformationally rich free-energy landscape. We have developed experimental kinetic and spectroscopic tools by using hydrostatic pressure to explore this free-energy landscape. The crystal structure of the N189A mutant enzyme in complex with the unreactive coenzyme analogue NADH(4) indicates that the nicotinamide moiety of the analogue is conformationally less restrained than the corresponding structure of the wild-type NADH(4) complex. This increased degree of conformational freedom in the N189A enzyme gives rise to the concept of multiple reactive configurations (MRCs), and we show that the relative population of these states across the free-energy landscape can be perturbed experimentally as a function of pressure. Specifically, the amplitudes of individual kinetic phases that were observed in stopped-flow studies of the hydride transfer reaction are sensitive to pressure; this indicates that pressure drives an altered distribution across the energy landscape. We show by absorbance spectroscopy that the loss of charge-transfer character of the enzyme-coenzyme complex is attributed to the altered population of MRCs on the landscape. The existence of a conformationally rich landscape in the N189A mutant is supported by molecular dynamics simulations at low and high pressure. The work provides firm experimental and computational support for the existence of parallel pathways arising from multiple conformational states of the enzyme-coenzyme complex. Hydrostatic pressure is a powerful and general probe of multidimensional energy landscapes that can be used to analyse experimentally parallel pathways for enzyme-catalysed reactions. We suggest that this is especially the case following directed mutation of a protein, which can lead to increased population of reactant states that are essentially inaccessible in the free-energy landscape of wild-type enzyme.

Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

In chlorophyll biosynthesis, the light-activated enzyme protochlorophyllide oxidoreductase catalyzes trans addition of hydrogen across the C-17-C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). This unique light-driven reaction plays a key role in the assembly of the photosynthetic apparatus, but despite its biological importance, the mechanism of light-activated catalysis is unknown. In this study, we show that Pchlide reduction occurs by dynamically coupled nuclear quantum tunneling of a hydride anion followed by a proton on the microsecond time scale in the Pchlide excited and ground states, respectively. We demonstrate the need for fast dynamic searches to form degenerate "tunneling-ready" configurations within the lifetime of the Pchlide excited state from which hydride transfer occurs. Moreover, we have found a breakpoint at -27 degrees C in the temperature dependence of the hydride transfer rate, which suggests that motions/vibrations that are important for promoting light-activated hydride tunneling are quenched below -27 degrees C. We observed no such breakpoint for the proton-tunneling reaction, indicating a reliance on different promoting modes for this reaction in the enzyme-substrate complex. Our studies indicate that the overall photoreduction of Pchlide is endothermic and that rapid dynamic searches are required to form distinct tunneling-ready configurations within the lifetime of the photoexcited state. Consequently, we have established the first important link between photochemical and nuclear quantum tunneling reactions, linked to protein dynamics, in a biologically significant system.

Solvent Effects on Environmentally Coupled Hydrogen Tunnelling During Catalysis by Dihydrofolate Reductase from Thermotoga maritima

Protein motions may be perturbed by altering the properties of the reaction medium. Here we show that dielectric constant, but not viscosity, affects the rate of the hydride-transfer reaction catalysed by dihydrofolate reductase from Thermotoga maritima (TmDHFR), in which quantum-mechanical tunnelling has previously been shown to be driven by protein motions. Neither dielectric constant nor viscosity directly alters the kinetic isotope effect of the reaction or the mechanism of coupling of protein motions to tunnelling. Glycerol and sucrose cause a significant increase in the rate of hydride transfer, but lead to a reduction in the magnitude of the kinetic isotope effect as well as an extension of the temperature range over which "passive" protein dynamics (rather than "active" gating motions) dominate the reaction. Our results are in agreement with the proposal that non-equilibrium dynamical processes (promoting motions) drive the hydride-transfer reaction in TmDHFR.

Conformational Relaxation following Hydride Transfer Plays a Limiting Role in Dihydrofolate Reductase Catalysis

The catalytic cycle of an enzyme is frequently associated with conformational changes that may limit maximum catalytic throughput. In Escherichia coli dihydrofolate reductase, release of the tetrahydrofolate (THF) product is the rate-determining step under physiological conditions and is associated with an "occluded" to "closed" conformational change. In this study, we demonstrate that in dihydrofolate reductase the closed to occluded conformational change in the product ternary complex (E.THF.NADP (+)) also gates progression through the catalytic cycle. Using NMR relaxation dispersion, we have measured the temperature and pH dependence of microsecond to millisecond time scale backbone dynamics of the occluded E.THF.NADP (+) complex. Our studies indicate the presence of three independent dynamic regions, associated with the active-site loops, the cofactor binding cleft, and the C-terminus and an adjacent loop, which fluctuate into discrete conformational substates with different kinetic and thermodynamic parameters. The dynamics of the C-terminally associated region is pH-dependent (p K a < 6), but the dynamics of the active-site loops and cofactor binding cleft are pH-independent. The active-site loop dynamics access a closed conformation, and the accompanying closed to occluded rate constant is comparable to the maximum pH-independent hydride transfer rate constant. Together, these results strongly suggest that the closed to occluded conformational transition in the product ternary complex is a prerequisite for progression through the catalytic cycle and that the rate of this process places an effective limit on the maximum rate of the hydride transfer step.

Functional Coupling between the Kv1.1 Channel and Aldoketoreductase Kvβ1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.

Ring contraction and selective ring opening of naphthenic molecules for octane number improvement

Different catalytic strategies have been evaluated to maximize the production of non-aromatic compounds with high octane number, starting from naphthenic molecules, which are typically obtained from the saturation of aromatics. The research octane number (RON), the motor octane number (MON), and the specific volume of the product mixtures were evaluated in each case. The product distribution obtained on acidic and Ptcontaining zeolites was investigated in the temperature range 533‐563 K in the presence of hydrogen at a total pressure of 2 MPa. It was found that skeletal isomerization (ring contraction) was the primary reaction in both HYand Pt/HY catalysts. The presence of Pt was found to enhance the stability of the catalyst, but also greatly altered the distribution of RC products, enhancing 1,1-dimethylcyclopentane. This enhancement can be explained in terms of a higher rate of hydride transfer caused by the presence of the metal. Evaluation of the octane numbers of the product indicated that a mixture of RC products results in rather high RON, but the MON and specific volume were about the same as that of the feed. To improve MON and specific volume an Ir/SiO2 catalyst with high hydrogenolysis activity was added to realize the ring opening (RO). The combination of RC and RO was tested on physical mixtures and segregated beds of Pt/HYand Ir/SiO2 catalysts in order to optimize the production of the iso-alkanes with highest octane number. It was found that with segregated catalyst beds, a better control of the selective breaking of C‐C bonds of RC isomers can be achieved, which optimizes octane number and specific volume. # 2007 Elsevier B.V. All rights reserved.

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s(-1) at 25 degrees C) and deuterium (12 to 31 s(-1) at 25 degrees C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25 degrees C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of "barrier engineering" by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.