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 ...