Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.     

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.     

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.     

Role of Arg100 in the active site of adenosylcobalamin-dependent glutamate mutase

Arginine-100 is involved in recognizing the gamma carboxylate of the substrate in glutamate mutase. To investigate its role in substrate binding and catalysis, this residue was mutated to lysine, tyrosine, and methionine. The effect of these mutations was to reduce k(cat) by 120-320-fold and to increase K(m(apparent)) for glutamate by 13-22-fold; K(m(apparent)) for adenosylcobalamin is little changed by these mutations. Even at saturating substrate concentrations, no cob(II)alamin could be detected in the UV-visible spectra of the Arg100Tyr and Arg100Met mutants. However, in the Arg100Lys mutant cob(II)alamin accumulated to concentrations similar to wild-type enzyme, which allowed the pre-steady-state kinetics of adenosylcobalamin homolysis to be investigated by stopped-flow spectroscopy. It was found that homolysis of the coenzyme is slower by an order of magnitude, compared with wild-type enzyme. Furthermore, glutamate binding is significantly weakened, so much so that the reaction exhibits second-order kinetics over the range of substrate concentrations used. The Arg100Lys mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild-type enzyme is reacted with deuterated substrates; this suggests that homolysis is slowed relative to hydrogen abstraction by this mutation.     

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.     

Rearrangement of L-2-hydroxyglutarate to L-threo-3-methylmalate catalyzed by adenosylcobalamin-dependent glutamate mutase

Adenosylcobalamin-dependent enzymes catalyze a variety of chemically difficult isomerizations in which a nonacidic hydrogen on one carbon is interchanged with an electron-withdrawing group on an adjacent carbon. We describe a new isomerization, that of L-2-hydroxyglutarate to L-threo-3-methylmalate, involving the migration of the carbinol carbon. This reaction is catalyzed by glutamate mutase, but k(cat) = 0.05 s(-)(1) is much lower than that for the natural substrate, L-glutamate (k(cat) = 5.6 s(-)(1)). EPR spectroscopy confirms that the major organic radical that accumulates on the enzyme is the C-4 radical of L-2-hydroxyglutarate. Pre-steady-state kinetic measurements revealed that L-2-hydroxyglutarate-induced homolysis of AdoCbl occurs very rapidly, with a rate constant approaching those measured previously with glutamate and methylaspartate as substrates. These observations are consistent with the rearrangement of the 2-hydroxyglutaryl radical being the rate-determining step in the reaction. The slow rearrangement of the 2-hydroxyglutaryl radical can be attributed to the poor stabilization by the hydroxyl group of the migrating glycolyl moiety of the radical transiently formed on the migrating carbon. In contrast, with the normal substrate the migrating carbon atom bears a nitrogen substituent that better stabilizes the analogous glycyl moiety. These studies point to the importance of the functional groups attached to the migrating carbon in facilitating the carbon skeleton rearrangement.     

Pre-steady-state kinetic studies on the Glu171Gln active site mutant of adenosylcobalamin-dependent glutamate mutase

Glutamate-171 is involved in recognizing the amino group of the substrate in glutamate mutase. The effect of mutating this residue to glutamine on the ability of the enzyme to catalyze the homolysis of adenosylcobalamin has been investigated using UV-visible stopped-flow spectroscopy. Although Glu171 does not contact the coenzyme, the mutation results in the apparent rate constants for substrate-induced homolysis of the coenzyme that are slower by 7-fold and 13-fold with glutamate and methylaspartate, respectively, than those measured for the wild-type enzyme; furthermore, it weakens the binding of these substrates by approximately 50-fold and approximately 400-fold, respectively. These observations lend support to the idea that the enzyme may use substrate binding energy to accelerate homolysis of the coenzyme. The mutation also results in isotope effects on coenzyme homolysis that are much smaller than the very large effects observed when the wild-type enzyme is reacted with deuterated substrates. This observation is consistent with adenosylcobalamin homolysis being slowed relative to hydrogen abstraction from the substrate.     

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.     

Crystallization and preliminary diffraction data for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii

Pink crystals of methylmalonyl-CoA mutase from Propionibacterium shermanii, a coenzyme B12 (5'-deoxyadenosylcobalamin)-dependent enzyme, have been obtained by the hanging-drop method in two different forms. One form lies in the space group P21, with unit cell dimensions a = 122 A, b = 160 A and c = 90 A, with beta = 104 degrees (1 A = 0.1 nm). There are two alpha beta dimers in the asymmetric unit. The crystals diffract to 3.2 A resolution and are suitable for high resolution X-ray diffraction studies.     

Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri

A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/- 0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21.     

Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii.

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.     

A novel reaction between adenosylcobalamin and 2-methyleneglutarate catalyzed by glutamate mutase

We describe a novel reaction of adenosylcobalamin that occurs when adenosylcobalamin-dependent glutamate mutase is reacted with the substrate analogue 2-methyleneglutarate. Although 2-methyleneglutarate is a substrate for the closely related adenosylcobalamin-dependent enzyme 2-methyleneglutarate mutase, it reacts with glutamate mutase to cause time-dependent inhibition of the enzyme. Binding of 2-methyleneglutarate to glutamate mutase initiates homolysis of adenosylcobalamin. However, instead of the adenosyl radical proceeding to abstract a hydrogen from the substrate, which is the next step in all adenosylcobalamin-dependent enzymes, the adenosyl radical undergoes addition to the exo-methylene group to generate a tertiary radical at C-2 of methyleneglutarate. This radical has been characterized by EPR spectroscopy with regiospecifically (13)C-labeled methyleneglutarates. Irreversible inhibition of the enzyme appears to be a complicated process, and the detailed chemical and kinetic mechanism remains to be elucidated. The kinetics of this process suggest that cob(II)alamin may reduce the enzyme-bound organic radical so that stable adducts between the adenosyl moiety of the coenzyme and 2-methyleneglutarate are formed.     

On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri

The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.     

Hydrogen transfer in catalysis by adenosylcobalamin-dependent diol dehydratase.

Studies [bachovchin, W. W., et al. (1978) Biochemistry 17, 2218] of the mechanism of inactivation of adenosylcobalamin-dependent diol dehydratase have led to the development of a general method to describe the kinetics of a reaction pathway containing a reservoir of mobile hydrogen. Analysis by this method of catalytic rate measurements for mixtures of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol supports a mechanism involving an intermediate with three equivalent hydrogens, in which hydrogen transfer from this intermediate to product is the major rate-contributing step. Other results using tritium as a trace label [essenberg, M. K., et al. (1971) J. Am. Chem. Soc. 93, 1242] are considered in light of these deuterium isotope studies.     

Location of deuterium oxide solvent isotope effects in the glutamate dehydrogenase reaction.

Stopped flow studies of D2O kinetic solvent isotope effects on the reaction catalyzed by L-glutamate dehydrogenase reveal, in addition to several effects apparently attributable simply to pKa shifts, a 2-fold pH-independent effect on the velocity of the steady state oxidative deamination of L-glutamate by enzyme and NADP. Comparable pH-independent D2O kinetic solvent isotope effects are seen both in a transient phase of the reaction in which alpha-ketoglutarate is displaced by L-glutamate from an enzyme-NADPH-alpha-ketoglutarate (product) complex and in an analogous model reaction in which alpha-ketoglutarate is displaced by D-glutamate. These results suggest that alpha-ketoglutarate dissociation from an enzyme-NADPH-alpha-ketoglutarate complex is rate-limiting in the steady state.