Abstract
Glutamate mutase is one of several adenosylcobalamin-dependent 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.
Highlights
We have shown that in glutamate mutase homolysis of the cobalt-carbon bond and hydrogen abstraction from the substrate are kinetically coupled processes [10]
Inactivation of Glutamate Mutase by 2-KG—Our initial experiments focused on whether 2-KG or the hydrated form that predominates in solution acted as either a competitive inhibitor or an irreversible inhibitor of glutamate mutase. 2-KG did not appear to bind very tightly to the enzyme, as no inhibition of activity was observed at concentrations up to 1 mM; for comparison, the Km for L-glutamate is 0.5 mM
Intriguing is the ability of these enzymes to stabilize free radical species, formed through homolysis of AdoCbl, by an estimated 25–30 kcal/mol relative to their formation in free solution [18, 19]
Summary
We have shown that in glutamate mutase homolysis of the cobalt-carbon bond and hydrogen abstraction from the substrate are kinetically coupled processes [10]. 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].
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