Review Article Coenzyme-B12-Dependent Glutamate Mutase

Marsh, E. Neil G.

doi:10.1006/bioo.2000.1168

Cited by 68 publications

(55 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[1] To effect these transformations, the unique Co III ÀC bond of AdoCbl must be homolytically cleaved to produce cob(II)-alamin (CblC) plus 5'-deoxyadenosyl radical (AdoC). This Abstract: The degree to which the corrin ring portion of coenzyme B 12 can facilitate the H-atom-abstraction step in the glutamate mutase (GM)-catalyzed reaction of (S)-glutamate has been investigated with density functional theory. The crystal structure of GM identifies two possible orientations of the ribose portion of coenzyme B 12 .…”

Section: Introductionmentioning

confidence: 99%

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases

Durbeej

Sandala

Bucher

et al. 2009

Chemistry A European J

View full text Add to dashboard Cite

The degree to which the corrin ring portion of coenzyme B(12) can facilitate the H-atom-abstraction step in the glutamate mutase (GM)-catalyzed reaction of (S)-glutamate has been investigated with density functional theory. The crystal structure of GM identifies two possible orientations of the ribose portion of coenzyme B(12). In one orientation (A), the OH groups of the ribose extend away from the corrin ring, whereas in the other orientation (B) the OH groups, especially that involving O3', are instead directed towards the corrin ring. Our calculations identify a sizable stabilization amounting to about 30 kJ mol(-1) in the transition structure (TS) complex corresponding to orientation B (TS(B)CorIm). In the TS complex where the ribose instead is positioned in orientation A, no such effect is manifested. The observed stabilization in TS(B)CorIm appears to be the result of favorable interactions involving O3' and the corrin ring, including a C-HO hydrogen bond. We find that the degree of stabilization is not particularly sensitive to the Co-C distance. Our calculations show that any potential stabilization afforded to the H-atom-abstraction step by coenzyme B(12) is sensitive to the orientation of the ribose moiety.

show abstract

Section: Introductionmentioning

confidence: 99%

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases

Durbeej

Sandala

Bucher

et al. 2009

Chemistry A European J

View full text Add to dashboard Cite

show abstract

“…The substrate radical next undergoes rearrangement to give the product radical, which is then quenched by hydrogen transfer from 5'-deoxyadenosine to give the product and regenerate the 5'-deoxyadenosyl radical. Finally, recombination of the adenosyl radical and cob(II)alamin to reform the coenzyme completes the catalytic cycle.Our interest in the mechanisms by which enzymes generate free radicals, as exemplified by dependent glutamate mutase, [10] led us to undertake an extensive set of KIE measurements to examine how hydrogen abstraction from the substrate and coenzyme homolysis are coupled together. [11][12][13][14][15] KIE measurements using deuterium-and tritium-labeled substrates and coenzyme have proved especially informative probes of the key steps of Co À C bond homolysis and hydrogen atom abstraction from substrate.…”

mentioning

confidence: 99%

“…[5][6][7][8][9][10] Radicals are generated by homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob(II)alamin, a cobalt(II) intermediate, and the 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5'-deoxyadenosine and the substrate radical.…”

mentioning

confidence: 99%

Intrinsic Deuterium Kinetic Isotope Effects in Glutamate Mutase Measured by an Intramolecular Competition Experiment

et al. 2007

View full text Add to dashboard Cite

Kinetic isotope effects (KIEs) provide a powerful tool to interrogate transition states of both enzymic and non-enzymic reactions, [1][2][3][4] provided that one can measure the intrinsic KIE on the chemical step of interest, that is, the isotope effect undiminished by other isotope-insensitive steps that may contribute to the overall rate of reaction. For small molecules reacting in solution the KIE measured usually represents the intrinsic value; however, for enzymes this is seldom the case. Here we report the first measurement of the intrinsic KIE in an adenosylcobalamin enzyme (AdoCbl, coenzyme B 12 ) for hydrogen atom transfer from substrate to coenzyme, which is a key step in the mechanism of this class of enzymes. For the B 12 enzyme glutamate mutase the intrinsic deuterium KIE for hydrogen transfer from the substrate, (2S,3S)-3-methylaspartate, to 5'-deoxyadensosine is 4.1. This value is well within the semiclassical limit for a deuterium isotope effect and is much smaller than the anomalously large KIEs previously measured in other B 12 enzymes and non-enzymatic model reactions, which were attributed to extensive hydrogen tunneling.Glutamate mutase one is of a group of AdoCbl-dependent enzymes that catalyze unusual isomerization reactions that formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (Scheme 1). [5][6][7][8][9][10] Radicals are generated by homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob(II)alamin, a cobalt(II) intermediate, and the 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5'-deoxyadenosine and the substrate radical. The substrate radical next undergoes rearrangement to give the product radical, which is then quenched by hydrogen transfer from 5'-deoxyadenosine to give the product and regenerate the 5'-deoxyadenosyl radical. Finally, recombination of the adenosyl radical and cob(II)alamin to reform the coenzyme completes the catalytic cycle.Our interest in the mechanisms by which enzymes generate free radicals, as exemplified by dependent glutamate mutase, [10] led us to undertake an extensive set of KIE measurements to examine how hydrogen abstraction from the substrate and coenzyme homolysis are coupled together. [11][12][13][14][15] KIE measurements using deuterium-and tritium-labeled substrates and coenzyme have proved especially informative probes of the key steps of Co À C bond homolysis and hydrogen atom abstraction from substrate. Pre-steady-state measurements on a number of enzymes have shown that hydrogen abstraction is kinetically coupled to CoÀC bond homolysis, [11,[16][17][18] as evidenced by the appearance of a kinetic isotope effect on cobalt-carbon bond homolysis when the enzymes are reacted with deuterated substrates.This observation implies that the 5'-dA radical is a highenergy intermediate that only has a fleeting existence. Furthermore, the KIEs reported for several AdoCbl enzymes are extremely large (r...

show abstract

“…This enzyme has proven a useful system to investigate the principles by which enzymes generate reactive radical intermediates and harness them for catalysis of chemically difficult reactions (12,13). Both wild-type and mutant enzymes have been studied using a variety of spectroscopic and kinetic methods to gain mechanistic insights into this reaction (14)(15)(16)(17)(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

et al. 2006

View full text Add to dashboard Cite

We have investigated the reaction of glutamate mutase with the glutamate analog, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with K i = 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 well simulated by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co 2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical at ∼ 10 Å from the cobalt, and at a tilt angle of ∼ 70° 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 substrate. 2-thiolglutarate appears to react in a manner analogous to glutamate by first forming a thioglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfurstablized 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. Keywordsenzyme; coenzyme-B 12 ; free radicals; isomerization; substrate analog; EPR spectrometry Adenosylcobalamin 1 (Coenzyme B 12 , AdoCbl) is the coenzyme for a group of enzymes that catalyze unusual rearrangement or elimination reactions, as well as for class II ribonucleotide reductases. In these enzymes, the coenzyme serves as a masked form of the 5′-deoxyadenosyl radical that is generated through homolytic fission of the AdoCbl cobalt-carbon bond (1-6). An interesting and still poorly understood aspect of these reactions is how these enzymes catalyze homolysis of the coenzyme because the generation of free radicals from stable, closed shell molecules is energetically highly unfavorable.In all cases, homolysis of AdoCbl is tightly coupled to the formation of substrate-based radicals (or in the case of ribonucleotide reductase, a protein-based thiyl radical). The initially formed 5′-deoxyadenosyl radical is extremely unstable and has never been observed spectroscopically.

show abstract

Review Article Coenzyme-B12-Dependent Glutamate Mutase

Cited by 68 publications

References 48 publications

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases

Intrinsic Deuterium Kinetic Isotope Effects in Glutamate Mutase Measured by an Intramolecular Competition Experiment

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

Contact Info

Product

Resources

About

Review Article Coenzyme-B12-Dependent Glutamate Mutase

Cited by 68 publications

References 48 publications

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B12‐Dependent Mutases

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B12‐Dependent Mutases

Intrinsic Deuterium Kinetic Isotope Effects in Glutamate Mutase Measured by an Intramolecular Competition Experiment

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

Contact Info

Product

Resources

About

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases

On the Importance of Ribose Orientation in the Substrate Activation of the Coenzyme B₁₂‐Dependent Mutases