Pre-Steady-State Kinetic Investigation of Intermediates in the Reaction Catalyzed by Adenosylcobalamin-Dependent Glutamate Mutase

Chih, Hung Wei; Marsh, E. Neil G.

doi:10.1021/bi991064t

Cited by 45 publications

(64 citation statements)

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“…The 5'-dA formed in the reaction was recovered by reverse-phase HPLC. [26] The deuterium content of the 5'-dA was determined by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). The 5'-dA isolated from the reaction contains a mixture of isotopomers, with contributions from natural abundance 13 C, 15 N, 17 O, and 2 H isotopes in addition to any deuterium from the substrate.…”

mentioning

confidence: 99%

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

et al. 2007

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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...

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confidence: 99%

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

et al. 2007

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“…Many researchers have considered axial ligand transinfl uence, steric effects, structural strain, protein infl uence, in addition to others [47][48][49][50][51][52][53][54][55][56][57][58] as determinant factors in the Co-C cleavage process. The hydrogen transfer from the substrate to 5-deoxysdenosyl ligand has also been considered as a triggering factor of the Co-C bond breaking [59][60][61][62][63][64][65][66][67]. These suggestions have been largely unconfi rmed by subsequent data.…”

Section: The Mechanisms Of Methylcobalamin and Adenosylcobalamin Actimentioning

confidence: 99%

The Nature of the Co-C Bond Cleavage Processes in Methylcob(II)Alamin and Adenosylcob(III)Alamin

Spataru¹,

Fernández²

2016

ChemJMold

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Abstract. Unfortunately, there are still signifi cant disagreements between experimental and theoretical data of rate constants, energy barriers for Co-C bond cleavage process and coordination numbers of vitamin B 12 coenzyme species in spite of the remarkable efforts done by research community. Therefore, no grounded mechanisms for Co-C vitamin B 12 coenzyme bond breaking process and subsequent reactions have been found up to now. The infl uence of the mixing orbitals e.g. Pseudo-Jahn-Teller and similar effects on the reactions paths of bond-cleavage mechanisms of vitamin B 12 co-factors must be taken into account by utilizing multi-reference methods, in particular multiconfi gurational self-consistent fi eld (MCSCF) method. Then, the change in total energy along the normal coordinate Q for the stretching mode including Co-C and Co-N bonds in vitamin B 12 cofactors is expected due to a "vibronic" coupling term, which couples an excited state and ground state by a second order derivative potential-energy operator. The strong state mixing effect is expected to lead to low energy barriers and to Co-C and Co-N axial bond cleavage events in agreement with experimental data. Afterward, the updated mechanisms of vitamin B 12 bio-processes can be determined.Keywords: vitamin B 12 , mechanism, bio-catalysis, Pseudo-Jahn-Teller effect, DFT, MCSCF. Received: December 2015/ Revised fi nal: February 2016/ Accepted: February 2016 IntroductionVitamin B 12 cofactor is one of eight B vitamins and is involved in mammalian cellular metabolism, infl uencing amino acid and DNA synthesis [1] and hematopoiesis. The best known human physiological function of vitamin B 12 is its role in promoting normal health in the brain and nervous system. Vitamin B 12 anemia can cause such neurologic dysfunction as weakness, fatigue, light-headedness, rapid heartbeat, rapid breathing, pale skin color, bruising, and bleeding (including bleeding gums). The more severe vitamin B 12 anemia dysfunctions are characterized by tingling or numbness of the fi ngers and toes, diffi culty walking, mood changes, depression, memory loss, disorientation and, in most severe cases, dementia [2]. Vitamin B 12 defi ciency has even been considered in playing a role in the development of Alzheimer's disease [3][4][5][6][7].The vitamin B 12 cofactor contains a cobalt atom surrounded by an equatorial corrin ring. Covalently linked to the central atom is a dimethylbenzimidazole that occupies one of the axial coordination positions. The opposite coordinate is available for several ligands in bio-medium or in solution; however, known biologically active structures containing adenosyl-or methyl have been considered most often (Figure 1).

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“…This suggests that a step other than hydrogen transfer is partially rate limiting in the overall reaction. Rapid quench experiments have allowed the pre-steady-state kinetics of product formation to be examined and have shown that product release is not rate limiting in either direction (40). As discussed below, this leaves the rearrangement of the substrate radical as a relatively slow step responsible for the suppression of the isotope effects.…”

Section: Figmentioning

confidence: 99%

Review Article Coenzyme-B12-Dependent Glutamate Mutase

Marsh

2000

Bioorganic Chemistry

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Pre-Steady-State Kinetic Investigation of Intermediates in the Reaction Catalyzed by Adenosylcobalamin-Dependent Glutamate Mutase

Cited by 45 publications

References 24 publications

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

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

The Nature of the Co-C Bond Cleavage Processes in Methylcob(II)Alamin and Adenosylcob(III)Alamin

Review Article Coenzyme-B12-Dependent Glutamate Mutase

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