Pre-steady-state kinetic investigation of intermediates in the reaction catalyzed by adenosylcobalamin-dependent glutamate mutase

Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L -threo-3-methylaspartate. Rapid quench experiments have been performed to m easure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5 '-deoxyadenosine was formed, and shown to be sufficiently fast for this ste p to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously fo r cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, pr oviding further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. N o burst phase was observed with either substrate, indicating that product r elease does not limit the rate of catalysis in either direction. For the co nversion of glutamate to methylaspartate, a single chemical step appeared t o dominate the overall rate, whereas in the reverse direction a lag phase w as observed, suggesting the accumulation of an intermediate, tentatively as cribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically com petent. When combined with information from previous mechanistic studies, t hese results allow a qualitative foe energy profile to constructed for the reaction catalyzed by glutamate mutase.