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, a nd kinetics are the relationships of five residues, Asp(13), Glu(14), Lys(16), His(41), and Arg(131), to the catalytic function and structur al organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhi bit 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 w ild-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 mutation s on k(cat) are consistent with the putative roles assigned to Asp(13) (catalytic base), His(41) (catalytic acid), and Glu(14) (structural o rganization of the active site). The modest effect of the R131L mutati on on the binding of aspartate is also in harmony with recent crystall ographic investigations, which suggests that Arg(131) stabilizes the c onformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with signif icant changes brought about by the mutation of the corresponding lysin es in the P-loop of other GTP- and ATP-binding proteins. Crystallograp hic structures place Lys(16) in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at correspo nding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transitio n state of the phosphotransfer reaction, Lys(16) may stabilize the enz yme structurally, In addition, the modest loss of catalytic activity o f the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.