Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Journal of Biological Chemistry

1999

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Glutamate mutase is one of several adenosylcobalamindependent 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.Glutamate mutase catalyzes the reversible isomerization of L-glutamate and L-threo-3-methylaspartate (1-3). It is one of a group of enzymes that use adenosylcobalamin (AdoCbl), 1 a biologically active form of vitamin B 12 , to catalyze unusual 1,2-rearrangements in which an electron-withdrawing group is interchanged with a hydrogen atom on an adjacent carbon (4 -6) The migrating group may be -OH, -NH 2 , or, as in the case of glutamate mutase, a carbon-containing fragment so that a skeletal rearrangement is effected (Fig. 1). The role of AdoCbl as the source of carbon-based radicals, which are unmasked by homolysis of the coenzyme cobalt-carbon bond, is well established (7). Experiments with isotopically labeled substrates and coenzyme have demonstrated that for all the enzymes examined including glutamate mutase, 5Ј-deoxyadenosine acts as the intermediate carrier of the migrating hydrogen (8, 9).Recently, we have shown that in glutamate mutase homolysis of the cobalt-carbon bond and hydrogen abstraction from the substrate are kinetically coupled processes (10). Adenosyl radical can therefore only be formed as a transient high energy species, or may not be formed at all if coenzyme homolysis and hydrogen abstraction are truly concerted processes. Similar findings have been reported for the related AdoCbl-dependent enzyme, methylmalonyl-CoA mutase (11). These unexpected observations have focused our attention on the role of the substrate in initiating free radical formation. A further intriguing and poorly understood aspect of these reactions is how the enzyme controls the rearrangement of substrate-radical to product-radical.In principle, these steps might be probed with substrates that give rise ...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Reaction of the Substrate Analog 2-Ketoglutarate with Adenosylcobalamin-dependent Glutamate Mutase

Roymoulik

Chen

Journal of Biological Chemistry

1999

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“…The purification from recombinant E. coli of the engineered single subunit glutamate mutase protein, GlmES, which has been used in this and previous mechanistic studies, has been described previously (17 (18). [8][9][10][11][12][13][14] C]-adenosylcobalamin was synthesized using established protocols described by Brown et.…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Cheng

2006

Biochemistry

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Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer we previously used presteady state, rapid quench techniques to measure the α-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-3 H]-adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72 respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 ± 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling. Keywordsenzyme; coenzyme-B 12 ; protein-radical; isomerization; transition state; secondary isotope effects Glutamate mutase is one of a group of adenosylcobalamin 1 (AdoCbl, coenzyme B 12 ) dependent enzymes that catalyze unusual carbon skeleton isomerisations. These rearrangements formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (1-6). The initial steps of these reactions involve homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob (II)alamin and 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5-deoxyadenosine and substrate radical (or protein radical in the case of AdoCbl-dependent ribonucleotide reductase). These steps have been studied in some detail for several B 12 enzymes and in each case homolysis and hydrogen abstraction are found to be kinetically coupled (7-10), as evidenced by the appearance of a kinetic isotope

“…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]. Racemic threo-3-methylaspartate was used in these experiments as the D-isomer has previously been shown to be neither a substrate nor an inhibitor of the enzyme [4].…”

Section: Methodsmentioning

confidence: 99%

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

Patwardhan

Archives of Biochemistry and Biophysics

2007

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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...