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PubMed1999

Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights.

BACKGROUND: Glutamate mutase (Glm) equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. Catalysis proceeds with the homolytic cleavage of the organometallic bond of the cofactor to yield a 5'-desoxyadenosyl radical. This radical then abstracts a hydrogen atom from the protein-bound substrate to initiate the rearrangement reaction. Glm from Clostridium cochlearium is a heterotetrameric molecule consisting of two sigma and two epsilon polypeptide chains. RESULTS: We have determined the crystal structures of inactive recombinant Glm reconstituted with either cyanocobalamin or methylcobalamin. The molecule shows close similarity to the structure of methylmalonyl CoA mutase (MCM), despite poor sequence similarity between its catalytic epsilon subunit and the corresponding TIM-barrel domain of MCM. Each of the two independent B12 cofactor molecules is associated with a substrate-binding site, which was found to be occupied by a (2S,3S)-tartrate ion. A 1:1 mixture of cofactors with cobalt in oxidation states II and III was observed in both crystal structures of inactive Glm. CONCLUSIONS: The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction.

Clostridium↗

PubMed1985

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors.

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.

Adamantane↗

PubMed2002

Dissociative phosphoryl transfer in PEP mutase catalysis: structure of the enzyme/sulfopyruvate complex and kinetic properties of mutants.

The crystal structure of PEP mutase from Mytilus edulis in complex with a substrate-analogue inhibitor, sulfopyruvate S-pyr (K(i) = 22 microM), has been determined at 2.25 A resolution. Mg(II)-S-pyr binds in the alpha/beta barrel's central channel, at the C-termini of the beta-strands. The binding mode of S-pyr's pyruvyl moiety resembles the binding mode of oxalate seen earlier. The location of the sulfo group of S-pyr is postulated to mimic the phosphonyl group of the product phosphonopyruvate (P-pyr). This sulfo group interacts with the guanidinium group of Arg159, but it is not aligned for nucleopilic attack by neighboring basic amino side chains. Kinetic analysis of site directed mutants, probing the key active site residues Asp58, Arg159, Asn122, and His190 correlate well with the structural information. The results presented here rule out a phosphoryl transfer mechanism involving a double displacement, and suggest instead that PEP mutase catalysis proceeds via a dissociative mechanism in which the pyruvyl C(3) adds to the same face of the phosphorus from which the C(2)O departs. We propose that Arg159 and His190 serve to hold the phosphoryl/metaphosphate/phosphonyl group stationary along the reaction pathway, while the pyruvyl C(1)-C(2) bond rotates upon formation of the metaphosphate. In agreement with published data, the phosphoryl group transfer occurs on the Si-face of PEP with retention of configuration at phosphorus.

Animals↗

PubMed2003

Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate.

UDP-galactopyranose mutase is a flavoprotein which catalyses the interconversion of UDP-galactopyranose and UDP-galactofuranose. The enzyme is of interest because it provides the activated biosynthetic precursor of galactofuranose, a key cell wall component of many bacterial pathogens. The reaction mechanism of this mutase is intriguing because the anomeric oxygen forms a glycosidic bond, which means that the reaction must proceed by a novel mechanism involving ring breakage and closure. The structure of the enzyme is known, but the mechanism, although speculated on, is not resolved. The overall reaction is electrically neutral but a crypto-redox reaction is suggested by the requirement that the flavin must adopt the reduced form for activity. Herein we report a thermodynamic analysis of the enzyme's flavin cofactor with the objective of defining the system and setting parameters for possible reaction schemes. The analysis shows that the neutral semiquinone (FADH(*)) is stabilized in the presence of substrate and the fully reduced flavin is the anionic FADH(-) rather than the neutral FADH(2). The anionic FADH(-) has the potential to act as a rapid 1-electron donor/acceptor without being slowed by a coupled proton transfer and is therefore an ideal crypto-redox cofactor.

Bacterial Proteins↗

PubMed2006

Reaction of adenosylcobalamin-dependent glutamate mutase with 2-thiolglutarate.

We have investigated the reaction of glutamate mutase with the glutamate analogue, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with a Ki of 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5'-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was simulated well by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical approximately 10 A from the cobalt and at a tilt angle of approximately 70 degrees to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with the substrate. 2-Thiolglutarate appears to react in a manner analogous to that of glutamate by first forming a thiolglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfur-stabilized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme, suggesting it is too stable to undergo further steps in the mechanism at a detectable rate.

Cobamides↗

PubMed2007

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase.

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.

Deoxyadenosines↗

PubMed1998

Identification of the 4-glutamyl radical as an intermediate in the carbon skeleton rearrangement catalyzed by coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.

Carbon↗

PubMed1999

Pre-steady-state kinetic investigation of intermediates in the reaction catalyzed by adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate. Rapid quench experiments have been performed to measure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5'-deoxyadenosine was formed, and shown to be sufficiently fast for this step to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously for cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, providing further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. No burst phase was observed with either substrate, indicating that product release does not limit the rate of catalysis in either direction. For the conversion of glutamate to methylaspartate, a single chemical step appeared to dominate the overall rate, whereas in the reverse direction a lag phase was observed, suggesting the accumulation of an intermediate, tentatively ascribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically competent. When combined with information from previous mechanistic studies, these results allow a qualitative free energy profile to constructed for the reaction catalyzed by glutamate mutase.

Catalysis↗

PubMed2002

Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri.

2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered. In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4. In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4. In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4. In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl[2'-(2)H(1)]itaconate and (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate. The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene[2'-(2)H(1)]glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR. In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl[2'-(2)H(1)]itaconate over its equilibrium value was observed ("E-overshoot"). The E-overshoot was only 3-4% with (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical. The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical. It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.

Catalysis↗

PubMed2004

Photolysis and recombination of adenosylcobalamin bound to glutamate mutase.

Ultrafast spectroscopic measurements are used to determine the kinetics of homolysis and recombination for adenosylcobalamin bound in the active site of glutamate mutase. These are the first such measurements on an adenosylcobalamin-dependent enzyme. A short-lived intermediate is formed prior to formation of the cob(II)alamin radical. This intermediate was not observed upon photolysis of adenosylcobalamin in free solution. The intrinsic rate constant for geminate recombination for adenosylcobalamin bound to glutamate mutase is 1.08 +/- 0.10 ns-1, only 16% smaller than the rate constant measured in free solution, 1.39 +/- 0.06 ns-1, suggesting the protein does not greatly perturb the stability of the cobalt-carbon bond upon binding the coenzyme.

Cobamides↗

PubMed2005

Time-resolved measurements of the photolysis and recombination of adenosylcobalamin bound to glutamate mutase.

Femtosecond to nanosecond transient absorption spectroscopy is used to investigate the photolysis of 5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) bound to glutamate mutase. The photochemistry of AdoCbl is found to be inherently dependent upon the environment of the cofactor. Excitation of AdoCbl bound to glutamate mutase results in formation of a metal-to-ligand charge transfer intermediate state which decays to form cob(II)alamin with a time constant of 105 ps. This observation is in contrast to earlier measurements in water where the photohomolysis proceeds through an intermediate state in which the axial dimethylbenzimidazole ligand appears to have dissociated, and measurements in ethylene glycol where prompt bond homolysis is observed (Yoder, L. M.; Cole, A. G.; Walker, L. A., II; Sension, R. J. J. Phys. Chem. B 2001, 105, 12180-12188). The quantum yield for formation of stable radical pairs in the enzyme is found to be phi = 0.05 +/- 0.03, and the resulting intrinsic rate constants for geminate recombination and "cage escape" are 1.0 +/- 0.1 and 0.05 +/- 0.03 ns(-1), respectively. The rate constant for geminate recombination is 30% less than that observed for AdoCbl in water or ethylene glycol. This reduction is insufficient to account for the 10(12)-fold increase in the homolysis rate observed when substrate is bound to the protein. Finally, the protein provides a cage to prevent diffusive loss of the adenosyl radical; however, the ultimate yield for long-lived radicals is determined by the evolution from a singlet to a triplet radical pair as proposed for AdoCbl in ethylene glycol.

Binding Sites↗

PubMed2005

Synthesis of a carbasugar analogue of a putative intermediate in the UDP-galp-mutase catalyzed isomerization.

[reaction: see text] The synthesis of the carbasugar analogue of 1,4-anhydro-beta-d-galactopyranose, a proposed intermediate in the reaction catalyzed by uridine diphosphate-alpha-d-Galp mutase, in racemic form via Diels-Alder and Barton decarboxylation chemistry is reported. This compound was found not to inhibit the mutase from Mycobacterium tuberculosis, indicating that the enzyme does not possess a 1,4-anhydro-beta-d-galactopyranose binding pocket.

Catalysis↗

PubMed1995

Synthesis of 3-arsonopyruvate and its interaction with phosphoenolpyruvate mutase.

3-Arsonopyruvate was prepared in four steps from glycine. The arsenic-carbon bond was formed by a Meyer reaction between alkaline arsenite and 2-bromo-3-hydroxy-2-(hydroxymethyl)propionic acid; the 3-arsono-2-hydroxy-2-(hydroxymethyl) propionic acid formed was oxidized with periodate to give 3-arsonopyruvate. This proves to be an alternative substrate for phosphoenolpyruvate mutase, giving pyruvate, which was assayed using lactate dehydrogenase. The K(m) is 20 microM, similar to that observed for the natural substrate phosphonopyruvate (17 microM), whereas the kcat. of 0.01 s-1 was much lower than that for phosphonopyruvate (58 s-1). Arsonopyruvate competitively inhibited the action of the mutase on phosphonopyruvate.

Arsenicals↗

PubMed1999

Reaction mechanism of phosphoglucosamine mutase from Escherichia coli.

The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically required for the interconversion of glucosamine-6-phosphate and glucosamine-1-phosphate (an essential step in the pathway for cell-wall peptidoglycan and lipopolysaccharide biosyntheses) was purified to homogeneity and its kinetic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping-pong bi-bi mechanism. The dephosphorylated and phosphorylated forms of GlmM could be separated by HPLC and coupled MS showed that only one phosphate was covalently linked to the active site of the enzyme. The site of phosphorylation was clearly identified as Ser102 in the 445-amino acid polypeptide. GlmM was also capable of catalysing the interconversion of glucose-1-phosphate and glucose-6-phosphate isomers, although at a much lower (1400-fold) rate. Interestingly, the mutational change of the Ser100 to a threonine residue resulted in a 20-fold increase of the nonspecific phosphoglucomutase activity of GlmM, suggesting that the presence of either a serine or a threonine at this position in the consensus sequence of hexosephosphate mutases could be one of the factors that determines the specificity of these enzymes for either sugar-phosphate or amino sugar-phosphate substrates.

Amino Acid Sequence↗

PubMed1999

Structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium cochlearium.

Glutamate mutase (Glm) is an adenosylcobamide-dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S, 3S)-3-methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an alpha2beta2 tetramer, whose assembly is mediated by coenzyme B12. The smaller of the protein components, GlmS, has been suggested to be the B12-binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR-study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum, which has previously been investigated using NMR-spectroscopy. Both solution structures of the two Glm B12-binding subunits share striking similarities with that determined by crystallography for the B12-binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp-x-His-x-x-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12-binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B12-free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained. Important elements of the binding site only become structured upon binding B12; these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the coenzyme.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of the carbon-phosphorus bond-forming enzyme phosphoenolpyruvate mutase from the mollusk Mytilus edulis.

The enzyme phosphoenolpyruvate mutase was purified to homogeneity from the mollusk Mytilus edulis. The subunit size of the native homotetramer was determined to be 34,000 Da. The steady-state kinetic constants for catalysis of the conversion of phosphonopyruvate to phosphoenolpyruvate at pH 7.5 and 25 degrees C were measured at kcat = 34 s-1, phosphonopyruvate Km = 3 microM, and Mg2+ Km = 4 microM. The enzyme displayed a broad specificity for divalent metal ion activation; Co2+, Mn2+, Zn2+, and Ni2+ are activators, whereas Ca2+ is not. Analysis of the pH dependence of the Mg2+-activated mutase-catalyzed reaction of phosphonopyruvate revealed one residue that must be protonated (apparent pKa = 8.3) and a second residue that must be unprotonated (apparent pKa = 7.7) for maximal catalytic activity.

Amino Acid Sequence↗

PubMed1999

The reaction of the substrate analog 2-ketoglutarate with adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase is one of several adenosylcobalamin-dependent enzymes that catalyze unusual rearrangements that proceed through a mechanism involving free radical intermediates. The enzyme exhibits remarkable specificity, and so far no molecules other than L-glutamate and L-threo-3-methylaspartate have been found to be substrates. Here we describe the reaction of glutamate mutase with the substrate analog, 2-ketoglutarate. Binding of 2-ketoglutarate (or its hydrate) to the holoenzyme elicits a change in the UV-visible spectrum consistent with the formation of cob(II)alamin on the enzyme. 2-ketoglutarate undergoes rapid exchange of tritium between the 5'-position of the coenzyme and C-4 of 2-ketoglutarate, consistent with the formation of a 2-ketoglutaryl radical analogous to that formed with glutamate. Under aerobic conditions this leads to the slow inactivation of the enzyme, presumably through reaction of free radical species with oxygen. Despite the formation of a substrate-like radical, no rearrangement of 2-ketoglutarate to 3-methyloxalacetate could be detected. The results indicate that formation of the C-4 radical of 2-ketoglutarate is a facile process but that it does not undergo further reactions, suggesting that this may be a useful substrate analog with which to investigate the mechanism of coenzyme homolysis.

Cobamides↗

PubMed1994

A family of hexosephosphate mutases in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.

Amino Acid Sequence↗