Stephen Bearne

Dalhousie University | Dal · Department of Biochemistry and Molecular Biology

A host of metabolic enzymes reversibly self‐assemble to form membrane‐less, intracellular filaments under normal physiological conditions and in response to stress. Often, these enzymes reside at metabolic control points, suggesting that filament formation affords an additional regulatory mechanism. Examples include cytidine‐5′‐triphosphate (CTP) s...

Activity-based protein profiling has facilitated the study of the activity of enzymes in proteomes, inhibitor development, and identification of enzymes that share mechanistic and active-site architectural features. Since methyl acyl phosphate monoesters act as electrostatically selective anionic electrophiles for the covalent modification of nucle...

Mandelate racemase (MR) catalyzes the Mg2+-dependent interconversion of (R)- and (S)-mandelate by stabilizing the altered substrate in the transition state (TS) by ∼26 kcal/mol. The enzyme has been employed as a model to explore the limits to which the free energy of TS stabilization may be captured by TS analogues to effect strong binding. Herein,...

CTP synthases (CTPS) catalyze the de novo production of CTP using UTP, ATP, and l‐glutamine with the anticancer drug metabolite gemcitabine‐5′‐triphosphate (dF‐dCTP) being one of its most potent nucleotide inhibitors. To delineate the structural origins of this inhibition, we solved the structures of Escherichia coli CTPS (ecCTPS) in complex with C...

Racemases and epimerases have attracted much interest because of their astonishing ability to catalyze the rapid α-deprotonation of carbon acid substrates with high pKa values (∼13-30) leading to the formation of d-amino acids or various carbohydrate diastereomers that serve important roles in both normal physiology and pathology. Enzymatic assays...

Mandelate racemase (MR) catalyses the Mg ²⁺ -dependent interconversion of ( R )- and ( S )-mandelate. To effect catalysis, MR stabilizes the altered substrate in the transition state (TS) by approximately 26 kcal mol –1 (–Δ G tx ), such that the upper limit of the virtual dissociation constant of the enzyme-TS complex is 2 × 10 –19 M. Designing TS...

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health an...

Repurposing of antiviral drugs affords a rapid and effective strategy to develop therapies to counter pandemics such as COVID‐19. SARS‐CoV‐2 replication is closely linked to the metabolism of cytosine‐containing nucleotides, especially cytidine‐5′‐triphosphate (CTP), such that the integrity of the viral genome is highly sensitive to intracellular C...

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural pr...

Cytidine-5′-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination o...

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2, and to a lesser extent, the alphacoronavirus HCoV-229E. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting synthesis of full-le...

The enolase superfamily (ENS) has served as a paradigm for understanding how enzymes that share a conserved structure, as well as a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation), evolved from a common progenitor to catalyze mechanistically diverse reactions. Enzymes of the mandelate racemase (MR)-subgro...

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed indus...

Racemases and epimerases catalyze the reversible interconversion of enantiomers and epimers. Available structures reveal that those enzymes solely utilizing a 1,1‐proton transfer mechanism all bind their chiral carbon acid substrate pairs in a mirror‐image packing orientation with a pseudo‐mirror plane defined by three groups on the stereogenic cen...

Boronic acids have been successfully employed as inhibitors of hydrolytic enzymes. Typically, an enzymatic nucleophile catalyzing hydrolysis adds to the electrophilic boron atom forming a tetrahedral species that mimics the intermediate(s)/transition state(s) for the hydrolysis reaction. We show that para-substituted phenylboronic acids (PBAs) are...

Unlike most enzymes, which exhibit stereospecific substrate binding, racemases and epimerases bind and catalyze the reversible interconversion of enantiomeric and epimeric pairs of substrates. Over the past 15 years, a growing number of racemase and epimerase structures have been solved, furnishing insights into the nature of chiral recognition of...

A rational approach that may be applied to a broad class of enzyme-catalyzed reactions to design enzyme inhibitors affords a powerful strategy, facilitating the development of drugs and/or molecular probes of enzyme mechanisms. A strategy for the development of substrate-product analogues (SPAs) as inhibitors of racemases and epimerases is elaborat...

Many enzymes catalyze the abstraction of a proton from a carbon acid substrate to initiate a variety of reactions; however, the development of a complete quantitative description of enzyme-catalyzed heterolytic cleavage of a C–H bond remains a challenge to enzymologists. To determine the pKaC–H value for such substrates bound at the active site, re...

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate using a two-base mechanism with Lys 166 acting as the Brønsted base to abstract the α-proton from (S)-mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-mandelate. The roles of these amino acids are re...

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg²⁺, to catalyse the interconversion of the enantiomers of mandelate. Although the active site Mg²⁺ may be replaced by Mn²⁺, Co²⁺, or Nⁱ²⁺, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters Kmapp, kcatapp, and (kcat/Km...

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell l...

Glutamate racemases (GR) catalyze the racemization of d‐ and l‐glutamate, and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d‐homocysteic acid (d‐HCA), while l‐HCA is a poor substrate. This enantioselectivity arises because l‐HCA pert...

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either L-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in...

Mandelate racemase (MR) serves as a paradigm for our understanding of enzyme-catalyzed deprotonation of a carbon acid substrate. To facilitate structure-function studies on MR using non-natural amino acid substitutions, we engineered the Cys92Ser/Cys264Ser variant (dmMR) as a platform for introducing Cys residues at specific locations for subsequen...

While enzyme activity is often regulated by a combination of substrate/effector availability and quaternary structure, many cytosolic enzymes may be further regulated through oligomerization into filaments. CTP synthase (CTPS) forms such filaments - a process that is promoted by the product CTP. The CTP analogue and active chemotherapeutic metaboli...

Rational approaches for the design of enzyme inhibitors furnish powerful strategies for developing pharmaceutical agents and tools for probing biological mechanisms. A new strategy for the development of gem-disubstituted substrate-product analogues as inhibitors of racemases and epimerases is elaborated using α-methylacyl-coenzyme A racemase from...

l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using rec...

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg²⁺-dependent, 1,1-proton transfer reaction that racemizes (R)- and (S)-mandelate. MR shares a partial reaction (i.e., the metal ion-assisted, Brønsted base-catalyzed proton abstraction of the α-proton of carboxylic acid substrates) and structural features ((β/α)7β-barrel and N-terminal...

The LDA-induced coupling of 2′,3′,5′-O-protected uridine with diethyl chlorophosphate, during the synthesis of 6-phosphonouridine, is accompanied by the formation of an unexpected side product. LDA adds slowly to the C4 position of the 2′,3′,5′-O-protected uridine after the initial reaction with diethyl chlorophosphate. The presence of the phosphor...

Background: Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction (abstraction of the α-proton from a carboxylate substrate). While the catalytic machinery responsible for the deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different ov...

The cover picture shows the potent inhibition of the enzyme CTP synthase (yellow) by gemcitabine triphosphate, a metabolite of the chemotherapeutic drug gemcitabine. Gemcitabine is catabolized into dF-dUTP in vivo; however, CTP synthase catalyzes the recycling of dF-dUTP to gemcitabine triphosphate in a glutamine-dependent manner in vitro. Inhibiti...

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents.Inhibition by the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5′-triphosph...

Isothermal titration calorimetry has been used to determine the stoichiometry and first (K 1) and second (K 2) conditional stability constants, where applicable, for the formation of complexes of benzohydroxamate (BzH) with Mg2+, Mn2+, Co2+, and Ni2+ ions, and benzoylphosphonate with Ni2+ ions. Binding determinations were conducted in HEPES buffer...

2,2-Bis(4-isobutylphenyl)propanoyl-CoA and 2,2-bis(4-t-butylphenyl)propanoyl-CoA are rationally designed, gem-disubstituted substrate-product analogues that competitively inhibit α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis with Ki values of 16.9 ± 0.6 and 21 ± 4 μM, respectively, exceeding the enzyme's affinity for the substrat...

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Brønsted acid and base catalysts, respectively, in...

This letter to the editor comments on the communication, "A Better Magnetic Stir Bar Retreiver". © 2015 The American Chemical Society and Division of Chemical Education, Inc.

Mandelate racemase (MR), a member of the enolase superfamily, catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate. Several α-keto acids are modest competitive inhibitors of MR (e.g., mesoxalate (Ki = 1.8 ± 0.3 mM) and 3-fluoropyruvate (Ki = 1.3 ± 0.1 mM)); but, surprisingly, 3-hydroxypyruvate (3-HP) is an irreversible, ti...

CTP plays a central role in nucleotide and lipid biosynthesis and is synthesized de novo by the catalytic action of CTP synthase (CTPS). CTPS is an important modulator of lymphocyte proliferation and has been identified as a potential therapeutic target for the treatment of acute lymphoblastic leukemia. The cytidine analogue gemcitabine is used to...

Regioselective S-acylation of coenzyme A (CoA) is achieved under aqueous conditions using various aliphatic and aromatic carboxylic acids activated as their methyl acyl phosphate monoesters. Unlike many hydrophobic activating groups, the anionic methyl acyl phosphate mixed anhydride is more compatible with aqueous solvents, making it useful for con...

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent, 1,1-proton transfer that interconverts of the enantiomers of mandelate. Since trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl...

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino...

Gibbs energy profiles provide students with a visual representation of the energy changes that occur during enzyme catalysis, making such profiles useful as teaching and learning tools. Traditional kinetic topics, such as the effect of pH on enzyme activity, are often not discussed in terms of Gibbs energy profiles. Herein, the symbolism of Gibbs e...

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) fro...

Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of Gib...

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal...

CTP Synthase from Trypanosoma brucei (TbCTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of antiprotozoal agents. GTP activates glutamine-dependent CTP formation catalyzed by TbCTPS at concentrations below 0.2 mM, but inhibits this activity at concentrations above 0.2 mM. TbCTPS catalyzes ammonia-dependent...

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the enantiomers of mandelic acid and a variety of aryl- and heteroaryl-substituted mandelate derivatives, suggesting that β,γ-unsaturation is a requisite feature of substrates for the enzyme. We show that β,γ-unsaturation is not an absolute requirement for cata...

The anaerobic, Gram-negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. The organism plays a central bridging role between early and late colonizers within dental plaque and plays a protective role against reactive oxygen species. Using a two-dimensional gel electrophore...

An analogue of orotidine 5'-monophosphate (OMP), 6-phosphonouridine 5'-monophosphate is a competitive inhibitor of OMP decarboxylase from E. coli, binding with an affinity similar to that of OMP. Hence the active site is capable of stabilizing negative charge distributed out of the plane of the pyrimidine ring, consistent with the notion of ground...

Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pKa values. Purification of recombinant MR...

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS activity (IC(50)=0.16-0.58mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition (IC(50)=0.060-0.121mM). An intact purine rin...

alpha-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular...

Cofactor-independent glutamate racemases (GRs) that supply the d-glutamate required for biosynthesis of the peptidoglycan that encapsulates bacterial cells are attractive targets for the development of antibacterial drugs. Recombinant GR from Fusobacterium nucleatum (FnGR), a Gram-negative anaerobe involved in periodontal disease, was overproduced,...

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. We show that phosphonate and phorphoramidate 5′-bismethylene triphosphate intermediate analogues 3-6 inhibit CTPS activity, as do the multivalent nucleotide inhibitors 7-10. These results supp...

The butyrate-producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many gut microorganisms, F. varium is capable of fermenting both amino acids and glucose. Although F. varium has been implicated in beneficial as well as pathological bacterium-host interactions, its genome has not been sequenced. To obta...

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to r...

Cytidine-5'-triphosphate synthase (CTPS) catalyzes the formation of cytidine triphosphate (CTP) from glutamine, uridine 5'-triphosphate (UTP), and adenosine 5'-triphosphate (ATP). This reaction proceeds via formation of the high-energy intermediate UTP-4-phosphate (UTP-4-P). Stable analogues of UTP-4-P may be potent inhibitors of CTPS and useful as...

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of man...

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A str...

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 of catabolizing glucose as the main energy source via the Embden-Meyerhof-Parnas pathway. 2-DE analyses revealed that the apparent concentratio...

Cytidine triphosphate synthetase (CTPS) catalyzes the formation of cytidine triphosphate from glutamine, uridine-5'-triphosphate (UTP), and adenosine-5'-triphosphate. Inhibitors of CTPS are of interest because of their potential as therapeutic agents. One approach to potent enzyme inhibitors is to use analogues of high energy intermediates formed d...

The inhibition of a purified β-galactosidase from “Spartan” apples by flavonoids and related compounds has been studied with a fluorimetric assay using the substrate 4-methylumbelliferyl β-D-galactoside. Certain flavonoids, flavonoid glycosides, phenolic acids, and polyphenols (quercetin, fustin, rutin, p-coumaric acid, and catechol) gave classical...

Mandelate racemase (MR) catalyzes the 1,1-proton transfer that interconverts the enantiomers of mandelate. The transition state/intermediate analogues N-hydroxyformanilide (K-i = 2.79 +/- 0.19 mu M) and cupferron (K-i = 2.67 +/- 0.09 mu M) are identified as potent competitive inhibitors of MR. The pH-pK(i) profile indicates that MR can bind either...

A protected, unsymmetrical bismethylene triphosphate analogue was prepared by sequential Michaelis-Arbuzov reactions on ethyl bis(halomethyl)phosphinates. This species was monodeprotected at one of the terminal phosphonate groups in high yield. The resulting monodeprotected compound was used to achieve the first syntheses of the bismethylene tripho...

Cytidine 5′-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or l-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2′,3′-dialdehyde adenosine 5′-triphosphate (oATP). oATP irreversibly inact...

Mandelate racemase has been studied as a paradigm for enzyme-catalyzed abstraction of a proton from carbon acids with relatively high pKa values. 1,1-Diphenyl-1-hydroxymethylphosphonate is a substrate-intermediate-product analogue and is a modest competitive inhibitor of the enzyme (Ki=1.41+/-0.09 mM), suggesting that simultaneous binding of the tw...

CPD-N is a cytokine-inducible CPD (carboxypeptidase-D) isoform identified in rat Nb2 T-lymphoma cells. The prototypic CPD (180 kDa) has three CP domains, whereas CPD-N (160 kDa) has an incomplete N-terminal domain I but intact domains II and III. CPD processes polypeptides in the TGN (trans-Golgi network) but the Nb2 CPD-N is nuclear. The present s...

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electr...

Many enzymes catalyse the heterolytic abstraction of the alpha-proton from a carbon acid substrate. Gerlt and Gassman have applied Marcus formalism to such proton transfer reactions to argue that transition states for concerted general acid-general base catalysed enolization at enzyme active sites occur late on the reaction coordinate (J. Am. Chem....

The adsorption behaviour of the enzyme, mandelate racemase (MR), and its substrate, (S)-mandelic acid (MA), was studied at a polycrystalline Pt surface in pH 7.4 phosphate buffer at 294K using the electrochemical quartz crystal nanobalance (EQCN) technique of simultaneous cyclic voltammetry (CV) and frequency measurements. It was shown that the EQC...

Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J...

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM,...

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol. We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to eval...

Mandelate racemase catalyzes the interconversion of the enantiomers of mandelic acid. The enzyme binds the intermediate analogues (R)- and (S)-alpha-fluorobenzylphosphonate, and alpha,alpha-difluorobenzylphosphonate with 100-2500 times less affinity than it exhibits for (R,S)-alpha-hydroxybenzylphosphonate at pH 7.5. This apparent low affinity, rel...

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to i...

Mandelate racemase catalyzes the interconversion of the enantiomers of mandelic acid. The enzyme binds the intermediate analogues (R)- and (S)-α-fluorobenzylphosphonate, and α,α-difluorobenzylphosphonate with 100–2500 times less affinity than it exhibits for (R,S)-α-hydroxybenzylphosphonate at pH 7.5. This apparent low affinity, relative to that of...

This paper describes the inhibition of GlmS by several analogues of the cis-enolamine intermediate in an attempt to probe the structural requirements for potent inhibition of this enzyme. The energetic contribution of the 2-amino group to binding of the product and the cis-enolamine intermediate is determined. MATERIALS AND METHODS D-Arabinose, D-a...

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH(3) or L-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (re...

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied...

Beta-methylaspartase (EC 4.3.1.2) was purified 20-fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAE-Sepharose. The enzyme is dimeric, consisting of two identical 46 kDa...

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specifi...

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with tither ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specifi...

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate...

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate...

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of d-glucosamine 6-phosphate fromd-fructose 6-phosphate using l-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents...

Mandelate racemase (EC 5.1.2.2) catalyzes the interconversion of the two stereoisomers of mandelic acid. A fixed-time assay for the quantification of mandelate racemase activity has been developed. The assay involves enzymatic conversion of R-mandelate to S-mandelate (or the reverse reaction) followed by separation and detection of the substrate an...

Mandelate racemase (EC 5.1.2.2) catalyzes the abstraction of a proton from a carbon atom adjacent to a carboxylate function, a reaction which is kinetically and thermodynamically unfavorable. Proton NMR spectroscopy and polarimetry were used to measure the rates of deuterium incorporation into the alpha-position of mandelate and the racemization of...

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. N-I...

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. N-I...

Pyrroline-5-carboxylate, an intermediate in the biosynthesis and degradation of glutamate, proline, and ornithine, acts as a strong reversible inhibitor of glucosamine-6-phosphate synthase, competitive with respect to glutamine. Proton magnetic resonance spectroscopy shows that, under these conditions, pyrroline-5-carboxylate exists in rapid equili...