Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction, i.e., metal-assisted, Bro̷nsted base-catalyzed abstraction α-proton from carboxylate substrate to form an enol(ate) intermediate. Although catalytic machinery responsible for initial deprotonation reaction has been conserved, divergent evolution led numerous ENS members that catalyze different overall reactions. Using differential scanning calorimetry, we examined contribution acid-base...

Abstract The butyrate‐producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many microorganisms, F. capable fermenting both amino acids and glucose. Although has been implicated in beneficial as well pathological bacterium–host interactions, its genome not sequenced. To obtain a better understanding the metabolic processes associated with acid fermentation by , we used gel‐based proteomic approach to examine changes soluble proteome accompanying...

Mandelate racemase (EC 5.1.2.2) catalyzes the abstraction of a proton from carbon atom adjacent to carboxylate function, reaction which is kinetically and thermodynamically unfavorable. Proton NMR spectroscopy polarimetry were used measure rates deuterium incorporation into α-position mandelate racemization (R)-mandelate, after samples had been incubated at elevated temperatures. Using an Arrhenius plot, value free energy activation for exchange was calculated be 34.6 (±0.9) kcal/mol under...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGlutamate .gamma.-Semialdehyde as a Natural Transition State Analog Inhibitor of Escherichia coli Glucosamine-6-phosphate SynthaseStephen L. Bearne and Richard WolfendenCite this: Biochemistry 1995, 34, 36, 11515–11520Publication Date (Print):September 12, 1995Publication History Published online1 May 2002Published inissue 12 September 1995https://pubs.acs.org/doi/10.1021/bi00036a026https://doi.org/10.1021/bi00036a026research-articleACS...

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of d-glucosamine 6-phosphate fromd-fructose using l-glutamine as ammonia source. Because N-acetylglucosamine is an essential building block both bacterial cell walls and fungal wall chitin, enzyme a potential target for antibacterial antifungal agents. The most potent carbohydrate-based inhibitor GlmS reported to date 2-amino-2-deoxy-d-glucitol 6-phosphate, analogue putative cis-enolamine intermediate formed during catalysis....

A proteome survey and MS analysis were conducted to investigate glucose metabolism in Fusobacterium varium, a butyrate-producing constituent of the indigenous human gut microflora. The bacterium was capable catabolizing as main energy source via Embden-Meyerhof-Parnas pathway. 2-DE analyses revealed that apparent concentrations six identified glycolytic enzymes (pyruvate kinase, enolase, glucose-6-phosphate isomerase, phosphoglycerate triosephosphate glyceraldehyde-3-phosphate dehydrogenase)...

The anaerobic, Gram-negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. organism central bridging between early late colonizers within dental plaque protective against reactive oxygen species. Using two-dimensional gel electrophoresis mass spectrometry approach, we have annotated 78 proteins proteome F. subsp. identified those whose apparent intracellular concentrations change response to either O(2)- or...

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of enantiomers mandelate, stabilizing altered substrate in transition state by 26 kcal/mol relative to ground state. To understand origins this binding discrimination, we determined X-ray crystal structures wild-type MR complexed with two analogues putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, 2.2-Å resolution. Benzohydroxamate is shown be a reasonable mimic...

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as source nitrogen. When is substrate, GTP required an allosteric effector to promote catalysis. Escherichia coli synthase, overexpressed a hexahistidine-tagged form, was purified high specific activity use metal-ion-affinity chromatography. Unfused generated by enzymic removal hexahistidine tag, displayed identical that native enzyme and used study effect on inhibition...

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of two enantiomers mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders magnitude. The rates forward and reverse reactions catalyzed by wild-type enzyme sluggish mutant (N197A) have been studied in absence presence several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values kcat kcat/Km sucrose-containing solutions. value...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDicarboxylic acid bis(methyl phosphates): anionic biomimetic crosslinking reagentsRonald Kluger, Andrew S. Grant, Stephen L. Bearne, and Marcel R. TrachselCite this: J. Org. Chem. 1990, 55, 9, 2864–2868Publication Date (Print):April 1, 1990Publication History Published online1 May 2002Published inissue 1 April 1990https://pubs.acs.org/doi/10.1021/jo00296a056https://doi.org/10.1021/jo00296a056research-articleACS PublicationsRequest reuse...

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts enantiomers of mandelate. Because trifluorolactate is also a substrate MR, we anticipated replacing phenyl rings competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was potent competitive [Ki 27 ± 4 μM; cf. Km 1.2 mM for...

Mandelate racemase (MR) catalyzes the interconversion of enantiomers mandelic acid, stabilizing altered substrate in transition state by 26 kcal/mol relative to ground state. To understand origins this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing intermediate analogues were examined for their ability inhibit MR. Comparison competitive inhibition constants revealed that an α-hydroxyl function is required recognition ligand as analogue. Two analogues,...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEnzymic hydration of an olefin: the burden borne by fumaraseStephen L. Bearne and Richard WolfendenCite this: J. Am. Chem. Soc. 1995, 117, 37, 9588–9589Publication Date (Print):September 1, 1995Publication History Published online1 May 2002Published inissue 1 September 1995https://pubs.acs.org/doi/10.1021/ja00142a037https://doi.org/10.1021/ja00142a037research-articleACS PublicationsRequest reuse permissionsArticle Views294Altmetric-Citations32LEARN...

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of two enantiomers mandelic acid with remarkable proficiency, stabilizing altered substrate in transition state by approximately 26 kcal/mol. We have used a series analogues (glycolates) and intermediate (hydroxamates) to evaluate contribution hydrophobic cavity within enzyme's active site ligand binding. Free energy changes accompanying binding glycolate derivatives correlated well substituent constant pi...

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as source nitrogen. GTP acts an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent at concentrations >0.15 mM and NH(3)-dependent all concentrations. A structure-activity study a variety guanosine analogues revealed that only few were capable activating to varying degrees: approximately 6-thio-GTP > ITP 5'-tetraphosphate O(6)-methyl-GTP...

CTP synthase catalyses the ATP-dependent formation of from UTP using either NH(3) or L-glutamine as nitrogen source. GTP is required an allosteric effector to promote glutamine hydrolysis. In attempt identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region amino acid sequence (residues 102-118) within domain Escherichia coli synthase. Mutant K102A exhibited wild-type activity with respect and glutamine; however, R105A, D107A, L109A G110A...

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts enantiomers of mandelate. Crystal structures MR reveal phenyl group all ground-state ligands is located within a hydrophobic cavity, remote site proton abstraction. forms numerous electrostatic and H-bonding interactions with α-OH carboxyl groups substrate, suggesting these polar may remain relatively fixed in position during catalysis while free to move between two...