The reaction of the substrate analog 2-ketoglutarate with adenosylcobalamin-dependent glutamate mutase

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, an d 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 glutamat e mutase with the substrate analog, 2-ketoglutarate. Binding of 2-ketogluta rate (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-ke toglutarate 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 aero bic conditions this leads to the slow inactivation of the enzyme, presumabl y through reaction of free radical species with oxygen. Despite the formati on of a substrate-like radical, no rearrangement of 2-ketoglutarate to 3-me thyloxalacetate could be detected. The results indicate that formation of t he 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 a nalog with which to investigate the mechanism of coenzyme homolysis.