Enzymatic Catalysis of Proton Transfer at Carbon Atoms

Gerlt, J.A.

doi:10.1002/9783527611546.ch35

Cited by 3 publications

(19 citation statements)

References 85 publications

(69 reference statements)

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“…Additional enhancement can be attributed to the positive electrostatic environment in the CS active site exerted by nearby Arg residues. These findings are congruent with concepts, advanced by Gerlt, Gassman, Cleland, and Kreevoy (13), that hydrogen bonding deployed within an active site can explain the ability of enzymes to achieve kinetically competent enolate formation.…”

Section: Discussionsupporting

confidence: 90%

“…We have presented evidence that dethiacitryl-CoA is the product of the interaction of TpCS 3 OAA with dethiaacetyl-CoA. The failure to detect dethiacitryl-CoA formation from [2-13 C]OAA by 13 C NMR in PCS solutions was puzzling and might be due to intermediate-NMR chemical shift exchange regime effects (7). However, we now believe any dethiacitryl-CoA that is formed by PCS would be below the level of detection by this relatively insensitive method.…”

Section: Discussionmentioning

confidence: 96%

“…The main finding of this work is that a stable TpCS 3 dethiacitryl-CoA complex is formed in solution. The nonhydrolyzable condensation product dethiacitryl-CoA has not been detected in reaction mixtures analyzed by 13 C NMR (PCS) (7,3) or ESI-MS (TpCS) (S. A. Kerfoot, unpublished observations). However, dethiaacetyl-CoA has been shown to undergo the condensation reaction in the solid state: a crystal structure of TpCS crystallized in the presence of saturating concentrations of OAA and dethiaacetyl-CoA contains only dethiacitryl-CoA at the active site [Protein Data Bank (PDB) entry 2r9e (C. Lehmann et al, unpublished observations)].…”

mentioning

confidence: 99%

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The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

et al. 2009

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Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA, and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flow and steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active site residues, we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the non-hydrolysable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not pig heart CS.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 96%

mentioning

confidence: 99%

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The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

et al. 2009

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“…There is a growing body of evidence that optimal hydrogen bonding in enzyme active sites may offer a significant contribution to catalytic accelerations 71. The importance of hydrogen bonds in stabilizing the enolate anion formed from deprotonated carbon acids was originally recognized by Gerlt and Gassman72 and Cleland and Kreevoy 73.…”

Section: Discussionmentioning

confidence: 99%

“…There is a growing body of evidence that optimal hydrogen bonding in enzyme active sites may offer a significant contribution to catalytic accelerations. 71 The importance of hydrogen bonds in stabilizing the enolate anion formed from deprotonated carbon acids was originally recognized by Gerlt and Gassman 72 and Cleland and Kreevoy. 73 Strong support for the enhancement of hydrogen bond strength in an enzyme's active site was determined by the model studies of Herschlag, 74,75 in which this reduction in free energy was quantified as being worth 0.93 kcal/mol/∆pK a 74 for a single hydrogen bond.…”

Section: In Vitro and In Silico Mutageneses Reveal The Strength Andmentioning

confidence: 99%

Determinants of Catalytic Power and Ligand Binding in Glutamate Racemase

et al. 2009

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Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D-and Lglutamate and are important for viability in several Gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L-to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the Cα of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from Cα, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.

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Enzymatic Catalysis of Proton Transfer at Carbon: Activation of Triosephosphate Isomerase by Phosphite Dianion

2007

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More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the phosphodianion group of GAP [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by "carving up" GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This "two-part substrate" preserves both the alpha-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1 s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd = 53 muM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the "mobile loop" and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from alpha-carbonyl carbon.

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Enzymatic Catalysis of Proton Transfer at Carbon Atoms

Cited by 3 publications

References 85 publications

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

Determinants of Catalytic Power and Ligand Binding in Glutamate Racemase

Enzymatic Catalysis of Proton Transfer at Carbon: Activation of Triosephosphate Isomerase by Phosphite Dianion

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