Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Marsh, E. Neil G.; Ballou, David P.

doi:10.1021/bi980512e

Cited by 144 publications

(250 citation statements)

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“…To compare our experimentally-determined isotope effects with those predicted by the computationally-determined free energy profile, we used equations 5 and 6 with the data in [20][21][22][23][24][25], in reactions that proceed with a high degree of quantum tunneling. Given that the BSS-catalyzed reaction involves radical chemistry similar to the enzymes mentioned above, the possibility that the intrinsic deuterium isotope effects are indeed extremely large cannot be entirely excluded.…”

Section: Expressions For D V and D (V/k)mentioning

confidence: 99%

“…This is well within the range of turnover numbers expected for a "typical" enzyme, although significantly higher than the values of about 0.1 s −1 at 4 °C and 0.35 s −1 at 30 °C for k cat that we calculate from our experiments. However, given that the computationally-determined free energy profile includes no explicit knowledge of the protein environment and does not account for substrate binding/ product release or protein conformational changes, the numbers agree reasonably well.To compare our experimentally-determined isotope effects with those predicted by the computationally-determined free energy profile, we used equations 5 and 6 with the data in [20][21][22][23][24][25], in reactions that proceed with a high degree of quantum tunneling. Given that the BSS-catalyzed reaction involves radical chemistry similar to the enzymes mentioned above, the possibility that the intrinsic deuterium isotope effects are indeed extremely large cannot be entirely excluded.…”

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confidence: 99%

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Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Marsh

2006

Biochemistry

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The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate that is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radicalcontaining family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on V max and V max /K m when deuterated toluene is the substrate. At 30 °C the isotope effects are 1.7 ± 0.2 and 2.9 ± 0.1 on V max and V max /K m , respectively, at 4 °C they increase slightly to2.2 ± 0.2 and 3.1 ± 0.1 respectively. We compare these results with the theoretical isotope effects on V max and V max /K m that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.Keywords toluene metabolism; free radicals; enzyme kinetics; Tauera. aromatica; glycyl radical enzyme Toluene and related aromatic compounds represent an important class of pollutants that are long-lived in the environment and have relatively high water solubility. This is a particular problem in environments where oxygen is scarce, such as ground water supplies. It was initially thought that the only routes to degrade such compounds were through oxidative pathways that require molecular oxygen to functionalize otherwise inert aromatic hydrocarbons. However, the recent discovery in bacteria of anaerobic pathways for the degradation of aromatic compounds has aroused much interest, both for the potential they offer for bio-remediation in situations where oxygen is scarce, and because of the novel chemistry involved (1,2).Various denitrifying and sulfate-reducing bacteria such as Thauera aromatica and Desulfobacula toluolica are able to live on toluene as their sole source of carbon and energy under anaerobic conditions (3,4). The first step in this pathway is the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, as shown in scheme 1, in which hydrogen in the methyl group of toluene is retained in the product (5). This remarkable chemical transformation is catalyzed by benzylsuccinate synthase 1 (BSS). † This research was supported by a grant from the National Institutes of Health, GM 59227 to E.N.G.M. BSS as a member of the growing class of glycyl radical-containing enzymes that includes pyruvate formate-lyase (PFL) and anaerobic ribonucleotide reductase (6-11). EPR studies show that the resting enzyme harbors an organic radical similar to that observed in these enzymes (12)(13)(14). Like other glycyl radical enzymes, BSS is extremely oxygen sensitive, and exposure to air results in oxidative cleavage of the α subunit at the site of the presumed glycyl radical (6).We recently investigated the stereochemistr...

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Section: Expressions For D V and D (V/k)mentioning

confidence: 99%

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confidence: 99%

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Marsh

2006

Biochemistry

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“…Firstly, it appears from our earlier studies that the ability of the enzyme to catalyzed AdoCbl homolysis is impaired. For the wild-type enzyme the observed rate constant for AdoCbl homolysis was 80 s -1 with methylaspartate as substrate [22]. However, for the Arg100Lys mutant the observed rate constant for homolysis of AdoCbl was 7 s -1 with methylaspartate as substrate, more than ten-fold slower than the wild-type enzyme [26].…”

Section: Discussionmentioning

confidence: 86%

“…AdoCbl, Lglutamate, D,L-threo-3-methylasparate, leucine and dansyl chloride were purchased from the Sigma Chemical Company. The sources of other materials have been described previously [3,4,22,27] or were purchased from commercial suppliers. [5'-3 H]-AdoCbl was prepared enzymatically by using GlmES protein to exchange tritium into the coenzyme from 3 H-labelled glutamate in a slight modification of the procedure described previously [31].…”

Section: Methodsmentioning

confidence: 99%

“…The crystal structures of the enzyme complexed with substrate and coenzyme analogs have been determined at high resolution [11,12]. The enzyme has been the subject of extensive mechanistic studies, both from our laboratory and other groups [13][14][15][16][17][18][19][20][21][22]. From these studies a fairly detailed mechanistic description of the wild-type enzyme has emerged and is summarized in Fig.…”

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Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine

Patwardhan

Marsh

2007

Archives of Biochemistry and Biophysics

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Arginine 100 plays an important role in substrate recognition in adenosylcobalamin-dependent glutamate mutase. We have examined how the mutation of this residue to lysine affects the partitioning of tritium, incorporated at the exchangeable position of the coenzyme, between substrate and product. We find that partitioning of tritium back to the substrate predominates in the mutant enzyme, regardless of whether the reaction is run in the forward or reverse direction. This contrasts with the behavior of the wild-type enzyme in which tritium partitions equally between substrate and product, independent of the direction of the reaction. From this we conclude that the mutation significantly impairs the ability of the enzyme catalyze the rearrangement of substrate radical to product radical. The results illustrate the importance of electrostatic interactions in stabilizing free radical intermediates in this class of enzymes. Keywordsenzyme; coenzyme-B 12 ; protein-radical; isomerization; free energy profile; isotope effect; isotope partitioning Glutamate mutase (EC 5.4.99.1) catalyses the reversible interconversion of L-glutamate to Lthreo-3-methylaspartate as the first step in the fermentation of L-glutamate by various species of Clostridia [1][2][3][4]. It is one of a group of Adenosylcobalamin-(AdoCbl, coenzyme B 12 ) dependent isomerases that catalyze unusual isomerizations in which a hydrogen atom on one carbon atom is interchanged with an electron-withdrawing group on an adjacent carbon [5][6][7][8][9][10]. These rearrangements proceed through a mechanism involving free radical intermediates that are generated by homolysis of AdoCbl.Glutamate mutase provides an attractive system with which to study the phenomenon of radical-mediated enzymatic catalysis: both the substrates are small, stable molecules; the reaction is freely reversible, and the enzyme requires no cofactors other than AdoCbl [3]. The crystal structures of the enzyme complexed with substrate and coenzyme analogs have been determined at high resolution [11,12]. The enzyme has been the subject of extensive mechanistic studies, both from our laboratory and other groups [13][14][15][16][17][18][19][20][21][22]. From these studies a fairly detailed mechanistic description of the wild-type enzyme has emerged and is summarized in Fig. 1. Most of intermediates postulated in this mechanism have been experimentally verified and the rates with which they are formed and decay have been measured, allowing a qualitative free energy profile for the enzyme to be constructed.* Corresponding author FAX (734) 615 3790, e-mail nmarsh@umich.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process error smay be discovered which could affect t...

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Glutamate Mutase

Kratky¹,

Gruber²

2004

Handbook of Metalloproteins

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The 5′‐adenosylcobalamin–dependent glutamate mutase catalyzes the reversible and stereospecific equilibration of ( S )‐glutamate with (2S,3S)‐3‐methylaspartate through a radical mechanism, whose first step consists of homolysis of the cofactor's cobalt–carbon bond. The enzyme occurs in clostridia, where it catalyzes the first step of the fermentation of glutamate. The protein exists as a heterotetramer of composition ɛ 2 σ 2 (B 12 ) 2 , with each of the two B 12 cofactor molecules located between an ɛ and a σ peptide. There are two substrate‐binding sites per heterotetramer at a distance of about 6 Å from the B 12 cofactor. The σ peptide folds as an α/β domain and is very similar to the B 12 ‐binding domains of other B 12 ‐dependent enzymes. The ɛ subunit consists of a (β/α) 8 ‐barrel (TIM‐barrel) domain, whose one end packs against the ‘upper’ face of the B 12 cofactor, thus forming the active site cavity.

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Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Cited by 144 publications

References 22 publications

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine

Glutamate Mutase

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