COUPLING OF COBALT-CARBON BOND HOMOLYSIS AND HYDROGEN-ATOM ABSTRACTION IN ADENOSYLCOBALAMIN-DEPENDENT GLUTAMATE MUTASE

Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carb on skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of t he coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspa rtate and with use of stopped-flow spectroscopy to follow the formatio n of cob(II)alamin. Either substrate induces rapid formation of cob(II )alamin, which accumulates to account for about 25% of the total enzym e species in the steady state when substrate is saturating. Measuremen ts of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbo n bond by at least 10(12)-fold. Very large isotope effects on cob(II)a lamin formation, of 28 and 35, are observed with deuterated L-glutamat e and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to su bstrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations o n the enzyme. The magnitude of the isotope effects suggests that hydro gen tunneling may play an important role catalysis.