CARBOXYMETHYLATION OF MUTS-CYSTEINE-15 SPECIFICALLY INACTIVATES ADENOSYLCOBALAMIN-DEPENDENT GLUTAMATE MUTASE - EXAMINATION OF THE ROLE OF THIS RESIDUE IN COENZYME-BINDING AND CATALYSIS

The sensitivity of adenosylcobalamin (AdoCbl)-dependent glutamate muta se toward thiol-directed reagents has been investigated. Iodoacetate s pecifically alkylates one cysteine residue, Cys-15, in MutS with conco mitant irreversible loss of enzyme activity. Cys-15 lies between the c onserved residues Asp-14 and His-16, that are believed to coordinate c obalt to form a Co-His-Asp hydrogen-bonded ''triad'' when AdoCbl is bo und by the enzyme. Although inactive, carboxymethylated MutS still bou nd AdoCbl with only a 5-fold increase in apparent K-d. To determine wh ether Cys-15 plays an essential role in catalysis, it was mutated to s erine and to alanine. These mutants were active, but both exhibited de creased V-max and increased apparent K-m and K-d for AdoCbl. To mimic the effect of carboxymethylation, Cys15 was mutated to aspartate and, as an isosteric control, to asparagine, Neither of these mutants was a ctive: MutS-C15N bound AdoCbl approximately 10-fold weaker than wild t ype, whereas MutS-C15N bound AdoCbl over 100 times less strongly than wild type. The results demonstrate both coenzyme-binding and catalysis to be very sensitive to mutations at position 15 that could potential ly perturb the Co-His-Asp hydrogen-bonding network.