I. Roymoulik et al., The reaction of the substrate analog 2-ketoglutarate with adenosylcobalamin-dependent glutamate mutase, J BIOL CHEM, 274(17), 1999, pp. 11619-11622
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.