Abstract
Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, 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 Km of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (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 Arg131 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 Lys16 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, Lys16 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.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Escherichia coli strain XL1-Blue came from Stratagene, a site-directed mutagenesis kit from Amersham Corp., and restriction enzymes from Promega
We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally
It was observed that when the mutant and wild-type enzymes were exposed to 0.5 M urea for varied periods of time and assayed for activity, the K16Q enzyme was significantly less stable than its wild-type counterpart. These results suggest that the K16Q mutant is less stable than the wild-type protein, provided that one accepts enzyme activity as a criterion of stability
Summary
Materials—Escherichia coli strain XL1-Blue came from Stratagene, a site-directed mutagenesis kit from Amersham Corp., and restriction enzymes from Promega. The chemicals used in this study were obtained from Sigma, and pur AϪ strain H1238 was a gift from Dr B. Site-directed Mutagenesis—Recombinant DNA manipulation was performed using standard procedures [10]. The mutagenic primers in this study are 5Ј-TTTACCTTCGGCACCCCATT-3Ј (Asp-13 3 Ala), 5Ј-.
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