ROLE OF THE His-Asp PAIR IN GLUTAMATE MUTASE

Glutamate mutase comprises two protein subunits: MutE and MutS. We were the first to identify sequence similarities between MutS and other B12 enzymes indicating that it forms a conserved domain involved in binding B12 ( Marsh & Holloway 1992). In particular two residues, histidine-16 and aspartate-14 are invariant and were later shown to be involved in coordinating cobalt. We have mutated His-16 to glycine and Asp-14 to both asparagine and alanine, and investigated the effects of these mutations on coenzyme binding and catalysis. This work has been published in Biochemistry.

All the mutants are profoundly impaired in catalysis. Even at the highest concentrations of protein and B12 feasible, none of the mutant proteins exhibited any detectable glutamate mutase activity when the holo-enzyme was reconstituted in the usual spectroscopic assay. We therefore developed a radiolabelled assay using 14C-labelled glutamate as the substrate and following the incorporation radioactivity into mesaconate. Using this much more sensitive assay we were able to observe turn-over with all of the mutants and determine their steady-state kinetic properties.

The mutants exhibit similar kinetic properties, regardless of the position or nature of the mutation. The most noticable effect is that kcat is decreased by about 1000-fold when compared with wild type enzyme. The apparent Km for glutamate of the mutants is raised by only two to four-fold, indicating that glutamate binding is little affected by mutations to the histidine-aspartate pair. Similarly, the apparent Km for B12 does not change greatly. However, the binding of B12 to glutamate mutase is, kinetically, complicated and values for Km may not reflect the true dissociation constants for B12-binding. Experiments are in progress to measure the binding of B12 directly.

In summary, it appears that the Co-His-Asp triad plays an important role in the mechanism of glutamate mutase, and probably influences both coenzyme binding and cobalt-carbon bond homolysis.