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PubMed2003

Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling.

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

Alanine↗
PubMed1997

New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs.

In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.

Binding, Competitive↗
PubMed1996

How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 A resolution.

BACKGROUND: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an alphabeta heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA. RESULTS: Reported here is the crystal structure at 2 A resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B12-binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a (beta/alpha)8 TIM barrel domain. CONCLUSIONS: The histidine-cobalt distance is very long (2.5 A compared with 1.95-2.2 A in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal-carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain.

Binding Sites↗
PubMed2000

Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

Aerobiosis↗
PubMed1995

Structural and electronic similarity but functional difference in methylmalonyl-CoA mutase between coenzyme B12 and the analog 2',5'-dideoxyadenosylcobalamin.

The cofactor analog 2',5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [5'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The 1H and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl-CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1990

Monofunctional chorismate mutase from Bacillus subtilis: kinetic and 13C NMR studies on the interactions of the enzyme with its ligands.

The interaction of the monofunctional chorismate mutase from Bacillus subtilis with chorismate and prephenate has been studied kinetically and by NMR spectroscopy with 13C specifically labeled substrates. Prephenate dominates the population of enzyme-bound species, and the "off" rate constant (approximately 60 s-1) obtained from line-broadening experiments is close to the value of kcat for chorismate (50 s-1) determined kinetically. The calculated "on" rate constant for prephenate (8 x 10(5) M-1 s-1) is similar to the value of kcat/Km for chorismate (5 x 10(5) M-1 s-1). The kinetic parameters of the Bacillus mutase are remarkably insensitive to pH over a wide range and display no solvent isotope effect. These results suggest that the enzyme-catalyzed reaction may be encounter controlled (slowed from the diffusion limit by some feature of the enzyme's active site) and that kcat for chorismate is determined by the product off rate. There is now no evidence to suggest that the skeletal rearrangement on the enzyme surface occurs by a pathway other than a pericyclic process.

Bacillus subtilis↗
PubMed2005

Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by transverse relaxation optimized spectroscopy-nuclear magnetic resonance.

The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR spectroscopy. TROSY methods were employed to assign 98% of the backbone (1)H(N), (1)H(alpha), (15)N, (13)C', and (13)C(alpha) resonances as well as 86% of the side chain (13)C resonances of the 44 kDa trimeric enzyme at 20 degrees C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on (15)N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein segment and the bound ligand, providing support for the proposal that the C-terminus can serve as a lid for the active site, limiting diffusion into and out of the pocket and possibly imposing conformational control over substrate once bound. Other regions of the protein that experience substantial ligand-induced changes also border the active site or lie along the subunit interfaces, indicating that the enzyme adapts dynamically to ligands by a sort of induced fit mechanism. It is believed that the mutase-catalyzed chorismate-to-prephenate rearrangement is partially encounter controlled, and backbone motions on the millisecond time scale, as seen here, may contribute to the reaction barrier.

Bacillus subtilis↗
PubMed2006

Alternative pathways for radical dissipation in an active site mutant of B12-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure of the enzyme reveals that Y243 is in van der Waals contact with the methyl group of the substrate and suggests a possible role for it in the stereochemical control of the reaction. This hypothesis was tested by designing a molecular hole by replacing the phenolic side chain of Y243 with the methyl group of alanine. The Y243A mutation lowered the catalytic efficiency >(4 x 10(4))-fold compared to wild-type enzyme, the K(M)app for the cofactor approximately 4-fold, and the cob(II)alamin concentration under steady-state turnover conditions approximately 2-fold. However, the mutation did not appear to lead to loss of the stereochemical preference for the substrate. The Y243A mutation is expected to create a cavity and should, in principle, allow accommodation of bulkier substrates. To test this, we used ethylmalonyl-CoA and allylmalonyl-CoA as alternate substrates. Surprisingly, both analogues resulted in suicidal inactivation, albeit in an O(2)-dependent and O(2)-independent fashion, respectively. The inactivation by allylmalonyl-CoA was further investigated, and revealed formation of cob(II)alamin at an approximately 1.5-fold higher rate than with wild-type mutase under single-turnover conditions. Product analysis revealed a stoichiometric mixture of 5'-deoxyadenosine, aquocobalamin, and allylmalonyl-CoA. Taken together, these results are consistent with an internal electron transfer from cob(II)alamin to the substrate analogue radical. These studies serve to emphasize the fine control exerted by Y243 in the vicinity of the substrate to minimize radical extinction in side reactions.

Acyl Coenzyme A↗
PubMed2006

Temperature dependence of the structure of the substrate and active site of the Thermus thermophilus chorismate mutase E x S complex.

Molecular dynamics (MD) simulations of Thermus thermophilus chorismate mutase substrate complex (TtCM x S) have been carried out at 298 K, 333 K, and the temperature of optimum activity: 343 K. The enzyme exists as trimeric subunits with active sites shared between two neighboring subunits. Two features distinguish intersubunit linkages of the thermophilic and mesophilic enzyme Bacillus subtilis chorismate mutase substrate complex (BsCM x S): (i) electrostatic interactions by intersubunit ion pairs (Arg3-Glu40*/41, Arg76-Glu51* and Arg69*-Asp101, residues labeled with an asterisk are from the neighboring subunit) in the TtCM x S are not present in the structure of the BsCM x S; and (ii) replacement of polar residues with short and nonpolar residues in the interstices of the TtCM x S tighten the intersubunit hydrophobic interactions compared to BsCM x S. Concerning the active site, electrostatic interactions of the critically placed Arg6 and Arg63* with the two carboxylates of chorismate place the latter in a reactive conformation to spontaneously undergo a Claisen rearrangement. The optimum geometry at the active site has the CZ atoms of the two arginines 11 A apart. With a decrease in temperature, Arg63* moves toward Arg6 and the average conformation structure of chorismate moves further away from the reactive ground state conformation. This movement is due to the decrease in distance separating the electrostatic (in the main) and hydrophobic interacting pairs holding the two subunits together.

Amino Acid Substitution↗
PubMed1996

Tritium isotope effects in adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-CoA and succinyl-CoA. The rate of tritium loss from 5'-[3H]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium was completely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-CoA in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-CoA in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using 14C-labeled methylmalonyl-CoA as substrate, was kH/kT = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.

Acyl Coenzyme A↗
PubMed1999

Role of the dimethylbenzimidazole tail in the reaction catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase.

The recent structures of cobalamin-dependent methionine synthase and methylmalonyl-CoA mutase have revealed a striking conformational change that accompanies cofactor binding to these proteins. Alkylcobalamins have octahedral geometry in solution at physiological pH, and the lower axial coordination position is occupied by the nucleotide, dimethylbenzimidazole ribose phosphate, that is attached to one of the pyrrole rings of the corrin macrocycle via an aminopropanol moiety. In contrast, in the active sites of these two B12-dependent enzymes, the nucleotide tail is held in an extended conformation in which the base is far removed from the cobalt in cobalamin. Instead, a histidine residue donated by the protein replaces the displaced intramolecular base. This unexpected mode of cofactor binding in a subgroup of B12-dependent enzymes has raised the question of what role the nucleotide loop plays in cofactor binding and catalysis. To address this question, we have synthesized and characterized two truncated cofactor analogues: adenosylcobinamide and adenosylcobinamide phosphate methyl ester, lacking the nucleotide and nucleoside moieties, respectively. Our studies reveal that the nucleotide tail has a modest effect on the strength of cofactor binding, contributing approximately 1 kcal/mol to binding. In contrast, the nucleotide has a profound influence on organizing the active site for catalysis, as evidenced by the retention of the base-off conformation in the truncated cofactor analogues bound to the mutase and by their inability to support catalysis. Characterization of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscopy reveals a pH-sensitive step that titrates to a pKa of 7.32 +/- 0.19 that is significantly different from the pKa of 3.7 for dimethylbenzimidazole in free AdoCbl. In contrast, the truncated cofactors associate very rapidly with the enzyme at rates that are too fast to measure. Based on these observations, we propose a model in which the base-on to base-off conformational change is slow and is assisted by the enzyme, and is followed by a rapid docking of the cofactor in the active site.

Benzimidazoles↗
PubMed2001

A hybrid potential reaction path and free energy study of the chorismate mutase reaction.

We present a combination of two techniques--QM/MM statistical simulation methods and QM/MM internal energy minimizations--to get a deeper insight into the reaction catalyzed by the enzyme chorismate mutase. Structures, internal energies and free energies, taken from the paths of the reaction in solution and in the enzyme have been analyzed in order to estimate the relative importance of the reorganization and preorganization effects. The results we obtain for this reaction are in good agreement with experiment and show that chorismate mutase achieves its catalytic efficiency in two ways; first, it preferentially binds the active conformer of the substrate and, second, it reduces the free energy of activation for the reaction relative to that in solution by providing an environment which stabilizes the transition state.

Bacillus subtilis↗
PubMed2003

Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor.

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.

Alanine↗
PubMed2005

Mirror "base-off" conformation of coenzyme B12 in human adenosyltransferase and its downstream target, methylmalonyl-CoA mutase.

Human adenosyltransferase synthesizes coenzyme B12, for the target mitochondrial B12 enzyme, methylmalonyl-CoA mutase. It binds B12 in the "base-off" conformation in both the Co2+ and Co3+ oxidation states as revealed by UV-visible and EPR spectroscopy although it lacks the signature DXHXXG motif found in other B12 proteins that bind the cofactor in this conformation. The "base-off" conformation, which is rare at physiological pH, mirrors that in the target enzyme, methylmalonyl-CoA mutase, which utilizes the product, AdoCbl. However, the coordination environment for cobalt in the two proteins is distinct, which is reflected in an approximately 40-fold difference in their affinity for the cofactor.

Alkyl and Aryl Transferases↗
PubMed2004

Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field.

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.

Catalysis↗
PubMed1977

A comparison of the association of yeast phosphoglycerate mutase (EC2.7.5.3) with that of haemoglobin. An ultracentrifuge study.

1. Previous work showed that yeast phosphoglycerate mutase (EC 2.7.5.3) has a mol.wt. of between 107000 and 110000. Preliminary examination showed that at dilutions less than 0.1 g/1 the enzyme dissociated into its subunits. 2. This dissociation was quantitatively examined by both equilibrium and velocity centrifugation. 3. The mathematical analysis of the equilibrium records was tested against oxyhaemoglobin in a variety of ionic strengths and at two temperatures. 4. The estimated L2,4 (interaction coefficient) for oxyhaemoglobin generally agreed with published values except at 6 degrees C in 0.9 M-NaCl, when it was 2.5 times larger than the published value. 5. Statistical analysis of ultracentrifugal-equilibrium experiments showed that the predominant reaction for phosphoglycerate mutase was monomer in equilibrium tetramer, to give an L1,4 of 40.3+/-23.4 (S.D.)1(3)-g(-3) at 20 degrees C. Decreasing the temperature decreased the association to given an enthalpy of between 40 and 60kJ/mol. 6. Analysis of velocity experiments carried out with concentrations varying from 0.3 to 17 g/1 gave an L1,4 of 3111(3)-g(-3). Incorporating errors from estimating S20,w into the analysis showed that this estimate could range from 893 to 1421(3)-g(-3). 7. The concentration-dependence of S20,w was 0.95 litre-g-1 and s020,w for the tetramer was 66.9ps. 8. These results are discussed in relation to the activity of the enzyme.

Hemoglobins↗
PubMed1986

The subunit structure of methylmalonyl-CoA mutase from Propionibacterium shermanii.

5'-Deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase was purified to homogeneity from Propionibacterium shermanii by a simplified procedure. The native enzyme has an apparent Mr of 165,000, similar to the enzyme from other sources but larger than previously reported. It consists of two non-identical subunits, of Mr 79,000 and 67,000 respectively. The smaller subunit is apparently not a proteolytic fragment of the larger one. The final preparation usually contained some inactive mutase, bearing a tenaciously bound cobalamin species. This protein proved to be readily separable from apoenzyme by fast protein liquid chromatography on anion-exchange columns.

Amino Acids↗