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 β-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 γ-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.