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...

Hydrogen atom transfer reactions between substrate and coenzyme are a key mechanistic feature of all AdoCbl-dependent enzymes. For one of these enzymes, glutamate mutase, we have investigated whether hydrogen tunneling makes a significant contribution to the mechanism by examining the temperature-dependence of the deuterium kinetic isotope effect associated with hydrogen atom transfer from methylaspartate to the coenzyme. To do this we designed a novel intra-molecular competition experiment that allowed us to measure the intrinsic kinetic isotope effect, even though hydrogen transfer may not be rate determining. From the Arrhenius plot of the kinetic isotope effect, the ratio of the pre-exponential factors AH/AD was 0.17 ± 0.04 and the isotope effect on the activation energy, ΔEa(D – H) was 1.94 ± 0.13 kcal/mol. The results imply that significant degree of hydrogen tunneling occurs in glutamate mutase, even though the intrinsic kinetic isotope effects are well within the semi-classical limit and are much smaller than those measured for other AdoCbl enzymes and model reactions for which hydrogen tunneling has been implicated.

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.

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...