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Ruma Banerjee - One of the best experts on this subject based on the ideXlab platform.
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Assembly and protection of the radical enzyme, Methylmalonyl-CoA Mutase, by its chaperone.
Biochemistry, 2006Co-Authors: Dominique Padovani, Ruma BanerjeeAbstract:MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, Methylmalonyl-CoA Mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA Mutase catalyzes the isomerization of Methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by Methylmalonyl-CoA Mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the Mutase for the 5‘-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 ± 71 to 210 ± 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 ± 0.33 μM, while in the presence of guanosine 5‘(β−γ imino)triphosphate, a nonhydrolyzable analogue of...
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Quantum catalysis in B12-dependent Methylmalonyl-CoA Mutase: experimental and computational insights.
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2006Co-Authors: Ruma Banerjee, Agnieszka Dybala-defratyka, Piotr PanethAbstract:B12-dependent Methylmalonyl-CoA Mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of Methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt–carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, Methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 °C to 49.9 at 5 °C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt–carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt–carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.
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Energetics of interaction between the G-protein chaperone, MeaB, and B12-dependent Methylmalonyl-CoA Mutase.
The Journal of biological chemistry, 2006Co-Authors: Dominique Padovani, Tetyana Labunska, Ruma BanerjeeAbstract:Abstract MeaB is an auxiliary protein that supports the function of the radical B12-dependent enzyme, Methylmalonyl-CoA Mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to for complexation of MeaB (in the presence and absence of nucleotides) with Methylmalonyl-CoA Mutase (in the presence and absence of cofactor). The dissociation constant for binding of Methylmalonyl-CoA Mutase and MeaB ranges from 34 ± 4 to 524 ± 66 nm, depending on the combination of nucleotide and Mutase form. HoloMutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apoMutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between ∼4000 and 8600A2 of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (Kd of 7.3 ± 1.9 and 6.2 ± 0.7 μm, respectively at 20 °C) and has low intrinsic GTPase activity (∼0.04 min–1 at 37 °C), which is stimulated ∼100-fold by Methylmalonyl-CoA Mutase. These studies provide insights into the energetics of interaction between the radical enzyme Methylmalonyl-CoA Mutase and MeaB, which are discussed.
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Alternative Pathways for Radical Dissipation in an Active Site Mutant of B12-dependent Methylmalonyl-CoA Mutase
Biochemistry, 2006Co-Authors: Dominique Padovani, Ruma BanerjeeAbstract: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 × 104)-fold compared to wild-type enzyme, the KMapp for the cofactor ∼4-fold, and the cob(II)alamin concentration under steady-state turnover conditions ∼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 ...
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Computational insights into the mechanism of radical generation in B12-dependent Methylmalonyl-CoA Mutase.
Journal of the American Chemical Society, 2006Co-Authors: Renata A. Kwiecień, Ruma Banerjee, Ilja V. Khavrutskii, Djamaladdin G. Musaev, Keiji Morokuma, Piotr PanethAbstract:ONIOM calculations have provided novel insights into the mechanism of homolytic Co−C5‘ bond cleavage in the 5‘-deoxyadenosylcobalamin cofactor catalyzed by Methylmalonyl-CoA Mutase. We have shown that it is a stepwise process in which conformational changes in the 5‘-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co−C5‘ bond elongates by ∼0.5 A from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is ∼10 kcal/mol, and the radical products are ∼2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5‘-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, Methylmalonyl-CoA.
Fred D Ledley - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis for dysfunction of some mutant forms of Methylmalonyl-CoA Mutase: deductions from the structure of methionine synthase.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Catherine L. Drennan, Fred D Ledley, Wayne A. Fenton, Rowena G. Matthews, David S. Rosenblatt, Martha L. LudwigAbstract:Abstract Inherited defects in the gene for Methylmalonyl-CoA Mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable Mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the Methylmalonyl-CoA Mutase. This portion of the Methylmalonyl-CoA Mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human Methylmalonyl-CoA Mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the Mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.
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Expression of recombinant human Methylmalonyl-CoA Mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.
Biochemical medicine and metabolic biology, 1993Co-Authors: E R Andrews, Ruud Jansen, Ana Crane, S. Cholin, Donald P. Mcdonnell, Fred D LedleyAbstract:Methylmalonyl-CoA Mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of Methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human Methylmalonyl-CoA Mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.
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Correction of Methylmalonyl-CoA Mutase deficiency inMut ^ o fibroblasts and constitution of gene expression in primary human hepatocytes by retroviral-mediated gene transfer
Somatic Cell and Molecular Genetics, 1992Co-Authors: Takako Sawada, Fred D LedleyAbstract:Methylmalonic acidemia is an often fatal inborn error of organic acid metabolism due to deficiency of Methylmalonyl-CoA Mutase. The cloning of genes encoding this enzyme and the advent of technologies for gene transfer have introduced the possibility of somatic gene therapy for this disorder. Gene therapy may require replacement of the defective enzyme in hepatocytes, which have a greater capacity for propionate metabolism than other somatic cells and represent the principle physiological site of propionate metabolism. We describe construction of an amphotropic retroviral vector containing the human Methylmalonyl-CoA Mutase cDNA. This vector is shown to transduce primary MCM-deficient fibroblasts and restore levels of [^14C]propionate metabolism by cultures of nonselected cells to normal. This vector will transduce primary human hepatocytes and direct transcription of recombinant human MCM from the integrated provirus. This work demonstrates the feasibility of retroviral-mediated gene transfer of Methylmalonyl-CoA Mutase into primary human cells, including hepatocytes which represent a difficult, but potentially necessary, target for gene therapy of methylmalonic acidemia.
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Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA Mutase: Implications for somatic gene therapy
Somatic Cell and Molecular Genetics, 1992Co-Authors: Michael Wilkemeyer, Jozsef Stankovics, Thomas Foy, Fred D LedleyAbstract:Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA Mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit >10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.
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Phenotype of disease in three patients with identical mutations in methylmalonyl CoA Mutase
Human Genetics, 1992Co-Authors: Ana Maria Crane, Laura S. Martin, David Valle, Fred D LedleyAbstract:We have previously identified a mutation in the gene for methylmalonyl CoA Mutase in a patient with the mut^- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.
Peter F. Leadlay - One of the best experts on this subject based on the ideXlab platform.
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Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase
Biochemistry, 2000Co-Authors: Nicolas H. Thoma, Philip R. Evans, Peter F. LeadlayAbstract: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, kcat was lowered by 102- and 103-fold for the His244Gln and His244Ala mutants, respectively, while the Km for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (kH/kT) for t...
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Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase†,‡
Biochemistry, 1998Co-Authors: Nicolas H. Thoma, Thomas W. Meier, Philip R. Evans, Peter F. LeadlayAbstract:The adenosylcobalamin-dependent Methylmalonyl-CoA Mutase catalyzes the reversible rearrangement of Methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of Methylmalonyl-CoA Mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among Methylmalonyl-CoA Mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5‘-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally...
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Tritium isotope effects in adenosylcobalamin-dependent Methylmalonyl-CoA Mutase.
Biochemistry, 1996Co-Authors: Thomas W. Meier, Nicolas H. Thoma, Peter F. LeadlayAbstract: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...
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Homology modeling of human Methylmalonyl-CoA Mutase: a structural basis for point mutations causing methylmalonic aciduria.
Protein science : a publication of the Protein Society, 1996Co-Authors: Nicolas H. Thoma, Peter F. LeadlayAbstract:Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent Methylmalonyl-CoA Mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric Methylmalonyl-CoA Mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii Methylmalonyl-CoA Mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the Mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of Methylmalonyl-CoA Mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.
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The synthetic substrate succinyl(carbadethia)-CoA generates cob(II)alamin on adenosylcobalamin-dependent Methylmalonyl-CoA Mutase.
Biochemical Journal, 1993Co-Authors: N H Keep, Gerry A. Smith, Michael C.w. Evans, G P Diakun, Peter F. LeadlayAbstract:Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent Methylmalonyl-CoA Mutase, has been prepared by a simplified procedure. When recombinant Mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-Methylmalonyl-CoA Mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by Methylmalonyl-CoA Mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.
János Rétey - One of the best experts on this subject based on the ideXlab platform.
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The putative coenzyme B12-dependent Methylmalonyl-CoA Mutase from potatoes is a phosphatase.
Bioorganic chemistry, 2008Co-Authors: Csaba Paizs, Tanja Diemer, János RéteyAbstract:Abstract The reported presence of a coenzyme B12-dependent Methylmalonyl-CoA Mutase in potatoes has been reexamined. The enzyme converting Methylmalonyl-CoA was purified to electrophoretic homogeneity. Examination of the reaction product by 1H, 31P NMR and mass spectrometry revealed that it was methylmalonyl-3′-dephospho-CoA. The phosphatase enzyme needs neither coenzyme B12 nor S-adenosylmethionine as a cofactor.
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Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction.
Bioorganic chemistry, 2000Co-Authors: Meinrad Kunz, János RéteyAbstract:Abstract An excellent substrate of Methylmalonyl-CoA Mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH 2 -CoA), was synthesized by a chemoenzymatic method and its α-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free Methylmalonyl-CoA Mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH 2 -CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (Methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH 2 -CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH 2 -CoA radical intermediate competing with the hydrogen transfer from 5′-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations.
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A base-off analogue of coenzyme-B12 with a modified nucleotide loop--1H-NMR structure analysis and kinetic studies with (R)-Methylmalonyl-CoA Mutase, glycerol dehydratase, and diol dehydratase.
European journal of biochemistry, 1997Co-Authors: László Poppe, William E. Hull, Erhard Stupperich, Thomas Buckel, János RéteyAbstract:(Co beta-5'-Deoxyadenosin-5'-yl)-(p-cresolyl)cobamide (Ado-PCC), an analogue of the base-off form of coenzyme-B12 (CoB12), was prepared by alkylation of (Co alpha/beta-cyano/aqua)-(p-cresolyl)cobamide (PCC) with 5'-chloro-5'-deoxyadenosine. The 500 MHz 1H-NMR spectrum of Ado-PCC in D2O at pH 7.4 was completely analyzed using COSY and NOESY two-dimensional experiments. The coenzyme and inhibitory activities of Ado-PCC were tested with three coenzyme-B12-dependent enzymes: (R)-Methylmalonyl-CoA Mutase, glycerol dehydratase, and diol dehydratase. Ado-PCC showed strong coenzyme activity with Methylmalonyl-CoA Mutase, which is known to bind the base-off form of CoB12. In contrast, Ado-PCC had no coenzyme activity but acted instead as a competitive inhibitor with glycerol dehydratase and diol dehydratase, which are likely to prefer the base-on form of CoB12. These results indicate that Ado-PCC, whose structure is analogous to the base-off form of CoB12, can be used for probing the mode of coenzyme binding by coenzyme-B12-dependent enzymes.
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Further insights into the mechanism of action of Methylmalonyl-CoA Mutase by electron paramagnetic resonance studies.
European journal of biochemistry, 1997Co-Authors: Andreas Abend, Valentin Illich, János RéteyAbstract:Novel analogues of Methylmalonyl-CoA and succinyl-CoA have been prepared and used for mechanistic investigations on the coenzyme-B12-dependent Methylmalonyl-CoA Mutase. 1-Carboxyethyl-CoA (1) and 2-carboxyethyl-CoA (2) as well as their sulphoxides (3 and 4) were moderately good inhibitors with Ki values 4–20 times higher than the Km for succinyl-CoA. 2-Carboxyethyl-CoA (2) and its sulphoxide 4 induced EPR signals when bound to the enzyme–coenzyme-B12 complex. The EPR spectrum of 2 and its sulphoxide 4 differed very much from those induced by the other substrates. In the case of 2 the EPR spectrum of the holoenzyme/inhibitor complex showed the presence of an organic radical coupled to cobal(II)amin. The same experiment with 4 leads to the formation of enzyme-bound cobal(II)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher Ki values and did not induce EPR signals binding to the enzyme-coenzyme-B12 complex. Formyl-CoA and acrylate inhibited the enzyme synergistically but were unable to induce EPR signals and to form the product. Ethylmalonyl-CoA, known as a poor substrate, induced a similar but less intense EPR signal than the natural substrate Methylmalonyl-CoA. The results are discussed in terms of the mechanism of the Methylmalonyl-CoA Mutase reaction.
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Electron Paramagnetic Resonance Studies of the Methylmalonyl-CoA Mutase Reaction
European journal of biochemistry, 1994Co-Authors: Ymin Zhao, Andreas Abend, Meinrad Kunz, Peter Such, János RéteyAbstract:The substrate-dependent homolysis of the cobalt-carbon bond and generation of organic radicals in the coenzyme-B12–Methylmalonyl-CoA-Mutase complex have been demonstrated by EPR measurements. Both the natural substrate Methylmalonyl-CoA, its 13C-substituted analogue and the non-hydrolysable synthetic substrates succinyl-dethia(carba)-CoA, succinyl-dethia(dicarba)-CoA and 4-carboxy-2-oxo-butyl-CoA induced similar but not identical EPR signals. 3-Carboxypropyl-CoA, a novel competitive inhibitor, has been synthesised. Its Ki, value of 89 ± 6 μM was in the same range as the Km, of succinyl-CoA. Using [5′-3H]adenosylcobalamin, an enzyme-dependent tritium transfer to the inhibitor has been shown. The enzyme-coenzyme-inhibitor complex also exhibited EPR signals that were less structured and less intensive than the corresponding signals with active substrates. These results prove that the inhibitor also induces cobalt-carbon bond homolysis and undergoes reversible hydrogen transfer but not rearrangement.
Philip R. Evans - One of the best experts on this subject based on the ideXlab platform.
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Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase
Biochemistry, 2000Co-Authors: Nicolas H. Thoma, Philip R. Evans, Peter F. LeadlayAbstract: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, kcat was lowered by 102- and 103-fold for the His244Gln and His244Ala mutants, respectively, while the Km for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (kH/kT) for t...
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Crystal structure of substrate complexes of Methylmalonyl-CoA Mutase.
Biochemistry, 1999Co-Authors: Filippo Mancia, G. A. Smith, Philip R. EvansAbstract:X-ray crystal structures of Methylmalonyl-CoA Mutase in complexes with substrate Methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme−substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5‘-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyrα89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-Methylmalonyl-CoA.
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Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase†,‡
Biochemistry, 1998Co-Authors: Nicolas H. Thoma, Thomas W. Meier, Philip R. Evans, Peter F. LeadlayAbstract:The adenosylcobalamin-dependent Methylmalonyl-CoA Mutase catalyzes the reversible rearrangement of Methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of Methylmalonyl-CoA Mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among Methylmalonyl-CoA Mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5‘-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally...
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Conformational changes on substrate binding to methylmalonyl CoA Mutase and new insights into the free radical mechanism.
Structure (London England : 1993), 1998Co-Authors: Filippo Mancia, Philip R. EvansAbstract:Abstract Background: Methylmalonyl CoA Mutase catalyses the interconversion of succinyl CoA and methylmalonyl CoA via a free radical mechanism. The enzyme belongs to a family of enzymes that catalyse intramolecular rearrangement reactions in which a group and a hydrogen atom on adjacent carbons are exchanged. These enzymes use the cofactor adenosylcobalamin (coenzyme B 12 ) which breaks to form an adenosyl radical, thus initiating the reaction. Determination of the structure of substrate-free methylmalonyl CoA Mutase was initiated to provide further insight into the mechanism of radical formation. Results: We report here two structures of methylmalonyl CoA Mutase from Propionibacterium shermanii . The first structure is of the enzyme in a nonproductive complex with CoA at 2.5 a resolution. This structure serves as a model for the substrate-free conformation of the enzyme, as it is very similar to the second much poorer 2.7 a resolution structure derived from a truly substrate-free crystal. The true substrate-free structure also shows the adenosyl group bound to the cobalt atom. Comparison of this structure with that of the previously reported complex of the enzyme with a substrate analogue shows that major conformational changes occur upon substrate binding. The substrate-binding site of the enzyme is located within a ( β α ) 8 TIM-barrel domain. In the absence of substrate, this TIM-barrel domain is split apart and the active site is accessible to solvent. When substrate binds, the barrel closes up with the substrate along its axis and the active site becomes completely buried. Conclusions: The closure of the active-site cavity upon substrate binding displaces the adenosyl group of the cofactor from the central cobalt atom into the active-site cavity. This triggers the formation of the free radical that initiates the rearrangement reaction. The TIM-barrel domain is substantially different from all others yet reported: in its unliganded form it is broken open, exposing the small hydrophilic sidechains which fill the centre. The typical barrel structure is only formed when substrate is bound.