Rearrangement of L-2-hydroxyglutarate to L-threo-3-methylmalate catalyzed by adenosylcobalamin-dependent glutamate mutase

Adenosylcobalamin-dependent enzymes catalyze a variety of chemically diffic ult isomerizations in which a nonacidic hydrogen on one carbon is interchan ged with an electron-withdrawing group on an adjacent carbon. We describe a new isomerization, that of L-2-hydroxyglutatrate to L-threo-3-methylmalate , involving the migration of the carbinol carbon. This reaction is catalyze d by glutamate mutase, but k(cat) = 0.05 s(-1) is much lower than that for the natural substrate, L-glutamate (k(cat) = 5.6 s(-1)). EPR spectroscopy c onfirms that the major organic radical that accumulates on the enzyme is th e C-4 radical of L-2-hydroxyglutarate. Pre-steady-state kinetic measurement s revealed that L-2-hydroxyglutarate-induced homolysis of AdoCbl occurs ver y rapidly, with a rate constant approaching those measured previously with glutamate and methylaspartate as substrates. These observations are consist ent with the rearrangement of the 2-hydroxyglutaryl radical being the rate- determining step in the reaction. The slow rearrangement of the 2-hydroxygl utaryl radical can be attributed to the poor stabilization by the hydroxyl group of the migrating glycolyl moiety of the radical transiently formed on the mi,orating carbon. In contrast, with the normal substrate the migratin g carbon atom bears a nitrogen substituent that better stabilizes the analo gous glycyl moiety, These studies point to the importance of the functional groups attached to the migrating carbon in facilitating the carbon skeleto n rearrangement.