HOW ENZYMES CONTROL THE REACTIVITY OF ADENOSYLCOBALAMIN - EFFECT ON COENZYME BINDING AND CATALYSIS OF MUTATIONS IN THE CONSERVED HISTIDINE-ASPARTATE PAIR OF GLUTAMATE MUTASE

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzy mes that catalyze unusual isomerizations that proceed through the form ation of radical intermediates. It shares a structurally similar cobal amin-binding domain with methylcobalamin-dependent methionine synthase . In particular, both proteins contain the ''DXHXXG'' cobalamin-bindin g motif, in which the histidine provides the axial ligand to cobalt, T he effects of mutating the conserved histidine and aspartate residues in methionine synthase have recently been described [Jarrett, J. T., A maratunga, M., Drennan, C. L., Scholten, J. D., Sands, R. H., Ludwig, M. L., & Matthews, R. G. (1996) Biochemistry 35, 2464-2475]. Here, we describe how similar mutations in the ''DXHXXG'' motif of glutamate mu tase affect coenzyme binding and catalysis in an adenosylcobalamin-dep endent reaction. The mutations made in the MutS subunit of glutamate m utase were His16Gly, His16Gln, Asp14Asn, Asp14Glu, and Asp14Ala. All t he mutations affect, in varying degrees, the rate of catalysis, the af finity of the protein for the coenzyme, and the coordination of cobalt . Mutations of either Asp14 or His16 decrease k(cat) by 1000-fold, and whereas cob(II)alamin accumulates as an intermediate in the wild-type enzyme, it does not accumulate in the mutants, suggesting the rate-de termining step is altered. The apparent Kd for adenosylcobalamin is ra ised by about 50-fold when His16 is mutated and by 5-10-fold when Asp 16 is mutated. There are extensive differences between the UV-visible spectra of wild-type and mutant holoenzymes, indicating that the mutan t enzymes coordinate cobalt less well. Overall, the properties of thes e mutants differ quite markedly from those observed when similar mutat ions were introduced into methionine synthase.