Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics

Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp(13), Glu(14), Lys(16), His(41), and Arg(131), to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the K-m of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and K-m values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg(131) to leucine caused only a 4-fold increase in the K-m for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on k(cat) are consistent with the putative roles assigned to Asp(13) (catalytic base), His(41) (catalytic acid), and Glu(14) (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg(131) stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins. Crystallographic structures place Lys(16) in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys(16) may stabilize the enzyme structurally, In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.