Solvent Deuterium Kinetic Isotope Effects Research Articles

“Stepwise or concerted or a hybrid mechanism for berberine bridge enzyme?”: A computational mechanistic study by QM-MM and QM methods

Berberine bridge enzyme (BBE) is a plant-based amine oxidase that catalyzes conversion of (S)-reticuline into (S)-scoulerine using flavin as cofactor in a stereospecific way in an alkaloid biosynthesis pathway. Based on the active site enzyme variants, a concerted mechanism was proposed involving hydride transfer, proton transfer, and substrate cyclization processes in a single step. In this mechanism, Glu417 residue acts as the catalytic base which deprotonates the phenolic proton of the substrate while a hydride ion transfers from the substrate to flavin and the substrate cyclizes. However, based on solvent and substrate deuterium kinetic isotope effect studies, it was proposed that the oxidation process occurs in a stepwise fashion in which a hydride ion transfer from substrate to flavin first, then cyclization of the substrate occurs together with the proton transfer process. In this study, we formulated computational models to elucidate the oxidation mechanism of (S)-reticuline into (S)-scoulerine using the crystal structure of enzyme complexed with (S)-reticuline. Both QM and QM-MM calculations revealed that a hybrid of concerted and stepwise mechanisms might be operative during the catalysis. It was found that a concerted hydride-proton transfer processes occurs forming a reactive intermediate which subsequently cyclize without an energy barrier as a decoupled step.

Catalytic Cycle of the Bifunctional Enzyme Phosphoribosyl-ATP Pyrophosphohydrolase/Phosphoribosyl-AMP Cyclohydrolase.

The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5'-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel.

Some differences in turnover kinetics of penams and cephems catalyzed by classes A and C ?-lactamases

The differences in the kinetic mechanism of catalysis of class A and class C ?-lactamases are shown by the manner by which these enzymes catalyze the hydrolyses of the penicillins and the cephalosporins. The hydrolysis of cephalosporins that are good substrates of the class C P99 ?-lactamase, where deacylation of an acyl-enzyme intermediate is rate-determining, has been shown to involve a free enzyme isoform (Adediran et al, 2021). We describe here how reconversion of this isoform to the native enzyme is accelerated by bases (e.g. imidazole) and salts (e. g. sodium chloride). The hydrolysis of penicillins by the P99 enzyme, where deacylation is also rate-determining, is not affected by imidazole and sodium chloride, a result that suggests that an enzyme isoform does not accumulate as an intermediate in turnover of this class of substrate. In support of these conclusions, solvent deuterium kinetic isotope effects on kcat values were changed by the presence of imidazole for turnover of cephalothin by the P99 enzyme but unaffected for benzylpenicillin turnover. The hydrolyses of cephalosporins and penicillins by the class A TEM-2 ?-lactamase were not affected by imidazole and sodium chloride and thus also may not involve an accumulating free enzyme isoform. Solvent deuterium kinetic isotope effects and proton inventories on the class A PC1 and P99 ?-lactamase-catalyzed hydrolyses of benzylpenicillin at saturating concentrations showed the deacylation transition states of these two classes of enzymes to be different with respect to proton motion.

Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition

Turnover of substrates by many enzymes involves free enzyme forms that differ from the stable form of the enzyme in the absence of substrate. These enzyme species, known as isoforms, have, in general, different physical and chemical properties than the native enzymes. They usually occur only in small concentrations under steady state turnover conditions and thus are difficult to detect. We show in this paper that in one particular case of an enzyme (a class C β-lactamase) with specific substrates (cephalosporins) the presence of an enzyme isoform (E′) can be detected by means of its different reactivity than the native enzyme (E) with a class of covalent inhibitors (phosphonate monoesters). Generation of E′ from E arises either directly from substrate turnover or by way of a branched path from an acyl-enzyme intermediate. The relatively slow spontaneous restoration of E from E′ is accelerated by certain small molecules in solution, for example cyclic amines such as imidazole and salts such as sodium chloride. Solvent deuterium kinetic isotope effects and the effect of methanol on cephalosporin turnover showed that for both E and E′, kcat is limited by deacylation of an acyl-enzyme intermediate rather than by enzyme isomerization.

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry.

The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD+:acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.

Insights into the Autoproteolytic Processing and Catalytic Mechanism of the Chlamydia trachomatis Virulence-Associated Protease CPAF.

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 μM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 μM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[2H4]-choline as a substrate. The limiting rate constants klim1 and klim2 at saturating substrate were well separated (klim1/klim2>9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that klim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that klim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the klim1 and klim2 values increased with increasing temperature, allowing for the analyses of H+ and H– transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the klim1 value (H2Oklim1/D2Oklim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the klim2 value gave lines with the slope(choline)>slope(D-choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.

Mechanism of the Novel Prenylated Flavin-Containing Enzyme Ferulic Acid Decarboxylase Probed by Isotope Effects and Linear Free-Energy Relationships.

Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the α═β bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(kcat/KM) for the reaction correlated well with the Hammett σ(-) parameter with ρ = -0.39 ± 0.03; r(2) = 0.93. The negative ρ value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin-product adduct through a cyclo-elimination reaction.

Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis

Saccharopine reductase catalyzes the reductive amination of l-α-aminoadipate-δ-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid–base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid–base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid–base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid–base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.

Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme

Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD+-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for the de novo synthesis of l-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys–Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of the Saccharomyces cerevisiae HIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008) Biochemistry47, 4169–4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized using initial rate studies. A decrease of 520-fold was observed in V and V/KMgHIc, suggesting the same step(s) limit the reaction at limiting and saturating MgHIc concentrations. Solvent kinetic deuterium isotope effects (SKIE) and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations. In addition, at limiting MgHIc, an inverse (SKIE) of 0.7 coupled to a significant normal effect of viscosogen (2.1) indicates equilibrium binding of MgHIc prior to the rate-limiting conformational change. The maximum rate exhibits a small partial change at high pH suggesting a pH-dependent conformational change, while V/KMgHIc exhibits the same partial change observed in V, and a decrease at low pH with a pKa of 6 reflecting the requirement for the unprotonated form of MgHIc to bind to enzyme. However, neither parameter reflects the pH dependence of the chemical reaction. This pH independence of the chemical reaction over the range 5.5–9.5 is consistent with the much slower conformational change that would effectively perturb the observed pK values for catalytic groups to lower and higher pH. In other words, the pH dependence of the chemical reaction will only be observed when chemistry becomes slower than the rate of the conformational change. Data support the hypothesis of the existence of a pre-catalytic conformational change coupled to the binding of MgHIc.

Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase.

The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ≤4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized D-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (k(red)) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on k(red). These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms.

Alcohol oxidation by flavoenzymes.

This review article describes the occurrence, general properties, and substrate specificity of the flavoenzymes belonging to the glucose-methanol-choline oxidoreductase superfamily and the l-α-hydroxyacid dehydrogenase family. Most of these enzymes catalyze the oxidations of hydroxyl groups, yielding carbonyl moieties. Over the years, carbanion, hydride transfer, and radical mechanisms have been discussed for these enzymes, and the main experimental evidences supporting these mechanisms are presented here. Regardless of the chemical nature of the organic substrate (i.e., activated and non-activated alcohols), a hydride transfer mechanism appears to be the most plausible for the flavoenzymes acting on CH-OH groups. The reaction of most of these enzymes likely starts with proton abstraction from the substrate hydroxyl group by a conserved active site histidine. Among the different approaches carried out to determine the chemical mechanisms with physiological substrates, primary substrate and solvent deuterium kinetic isotope effect studies have provided the most unambiguous evidences. It is expected that the numerous studies reported for these enzymes over the years will be instrumental in devising efficient industrial biocatalysts and drugs.

Loop‐loop interactions govern multiple steps in indole‐3‐glycerol phosphate synthase catalysis

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site β1α1 and β2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

Reactive thioglucoside substrates for β-glucosidase

A new, very efficient, class of thioglycoside substrates has been found for β-glucosidase. While thioglycosides are usually resistant to hydrolysis, even in the presence of acids or most glycohydrolases, the β-d-glucopyranosides of 2-mercaptobenzimidazole (GlcSBiz) and 2-mercaptobenzoxazole (GlcSBox) have been found to be excellent substrates for β-glucosidase from both sweet almond (a family 1 glycohydrolase) and Aspergillus niger (a family 3 glycohydrolase), reacting nearly as well as p-nitrophenyl β-d-glucoside. The enzyme-catalyzed hydrolysis of GlcSBiz proceeds with retention of configuration. As with the (1000-fold slower) hydrolysis of phenyl thioglucosides catalyzed by the almond enzyme, the pL (pH/pD)-independent kcat/KM does not show a detectable solvent deuterium kinetic isotope effect (SKIE), but unlike the hydrolysis of phenyl thioglucosides, a modest SKIE is seen on kcat [D2Okcat=1.28 (±0.06)] at the pL optimum (5.5⩽pL⩽6.6). A solvent isotope effect is also seen on the KM for the N-methyl analog of GlcSBiz. These results suggest that the mechanism for the hydrolysis of the β-thioglucoside of 2-mercaptobenzimidazole and of 2-mercaptobenzoxazole involves remote site protonation (at the ring nitrogen) followed by cleavage of the thioglucosidic bond resulting in the thione product.

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0.22 ± 0.03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1.8 ± 0.2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.

Trifunctional Metal Ion-Catalyzed Solvolysis: Cu(II)-Promoted Methanolysis of N,N-bis(2-picolyl) Benzamides Involves Unusual Lewis Acid Activation of Substrate, Delivery of Coordinated Nucleophile, Powerful Assistance of the Leaving Group Departure

The methanolyses of Cu(II) complexes of a series of N,N-bis(2-picolyl) benzamides (4a-g) bearing substituents X on the aromatic ring were studied under (s)(s)pH-controlled conditions at 25 °C. The active form of the complexes at neutral (s)(s)pH has a stoichiometry of 4:Cu(II):((-)OCH(3))(HOCH(3)) and decomposes unimolecularly with a rate constant k(x). A Hammett plot of log(k(x)) vs σ(x) values has a ρ(x) of 0.80 ± 0.05. Solvent deuterium kinetic isotope effects of 1.12 and 1.20 were determined for decomposition of the 4-nitro and 4-methoxy derivatives, 4b:Cu(II):((-)OCH(3))(HOCH(3)) and 4g:Cu(II):((-)OCH(3))(HOCH(3)), in the plateau region of the (s)(s)pH/log(k(x)) profiles in both CH(3)OH and CH(3)OD. Activation parameters for decomposition of these complexes are ΔH(++) = 19.1 and 21.3 kcal mol(-1) respectively and ΔS(++) = -5.1 and -2 cal K(-1) mol(-1). Density functional theory (DFT) calculations for the reactions of the Cu(II):((-)OCH(3))(HOCH(3)) complexes of 4a,b and g (4a, X = 3,5-dinitro) were conducted to probe the relative transition state energies and geometries of the different states. The experimental and computational data support a mechanism where the metal ion is coordinated to the N,N-bis(2-picolyl) amide unit and positioned so that it permits delivery of a coordinated Cu(II):((-)OCH(3)) nucleophile to the C═O in the rate-limiting transition state (TS) of the reaction. This proceeds to a tetrahedral intermediate INT, occupying a shallow minimum on the free energy surface with the Cu(II) coordinated to both the methoxide and the amidic N. Breakdown of INT is a virtually barrierless process, involving a Cu(II)-assisted departure of the bis(2-picolyl)amide anion. The analysis of the data points to a trifunctional role for the metal ion in the solvolysis mechanism where it activates intramolecular nucleophilic attack on the C═O group by coordination to an amidic N in the first step of the reaction and subsequently assists leaving group departure in the second step. The catalysis is very large; compared with the second order rate constant for methoxide attack on 4b, the computed reaction of CH3O(-) and 4b:Cu(II):(HOCH(3))(2) is accelerated by roughly 2.0 × 10(16) times.

Isotope Effects Suggest a Stepwise Mechanism for Berberine Bridge Enzyme

The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (S)-reticuline to (S)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on k(red), the rate constant for flavin reduction, is pH-independent, indicating that C-H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on k(red) are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived.

Transition State Analysis of Enolpyruvylshikimate 3-Phosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis

Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.

Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3-Phosphate (EPSP)

Proton transfer to carbon represents a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs) were measured for acid-catalyzed hydrolysis of the enol ether functionality of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog of the EPSP synthase (AroA) reaction. The large solvent deuterium KIE demonstrated that protonating C3 was the rate-limiting step, and the lack of solvent hydron exchange into EPSP demonstrated that protonation was irreversible. The reaction mechanism was stepwise, with C3, the methylene carbon, being protonated to form a discrete oxacarbenium ion intermediate before water attack at the cationic center, that is, an AH(‡)*AN (or AH(‡) + AN) mechanism. The calculated 3-(14)C and 3,3-(2)H2 KIEs varied as a function of the extent of proton transfer at the transition state, as reflected in the C3-H(+) bond order, nC3-H+. The calculated 3-(14)C KIE was a function primarily of C3 coupling with the movement of the transferring proton, as reflected in the reaction coordinate contribution ((light)ν(‡)/(heavy)ν(‡)), rather than of changes in bonding. Coupling was strongest in early and late transition states, where the reaction coordinate frequency was lower. The other calculated (14)C and (18)O KIEs were more sensitive to interactions with counterions and solvation in the model structures than nC3-H+. The KIEs revealed a moderately late transition state with significant oxacarbenium ion character and with a C3-H(+) bond order ≈0.6.

Supporting role of lysine 13 and glutamate 16 in the acid–base mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase (SDH) catalyzes the NAD+ dependent oxidative deamination of saccharopine to form lysine (Lys) and α-ketoglutarate (α-kg). The active site of SDH has a number of conserved residues that are believed important to the overall reaction. Lysine 13, positioned near the active site base (K77), forms a hydrogen bond to E78 neutralizing it, and contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate 16 is within hydrogen bond distance to the Nε atom of R18, which has strong H-bonding interactions with the α-carboxylate and α-oxo groups of α-kg. Mutation of K13 to M and E16 to Q decreased kcat by about 15-fold, and primary and solvent deuterium kinetic isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting for the overall reaction. The pH-rate profiles for K13M exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pKas of catalytic groups. Elimination of E16 affects optimal positioning of R18, which is involved in binding and holding α-kg in the correct conformation for optimum catalysis. In agreement, a ΔΔG°′ of 2.60kcal/mol is estimated from the change in Kα-kg for replacing E16 with Q.