Adenylosuccinate synthase (ASS) from Saccharomyces cerevisiae has been shown to bind specifically to the T‐rich side of the autonomously replicating sequence (ARS) core consensus sequence [Zeidler, R., Hobert, O., Johannes, L., Faulhammer, H. & Krauss, G. (1993) J. Biol. Chem. 268, 20191–20197]. We have cloned and sequenced the gene for ASS and have studied in detail the enzymatic properties and DNA‐binding activity of ASS. The deduced amino acid sequence of the yeast ASS is highly similar to the same enzymes from other sources from which it is however distinguished by its more basic nature. We show that the enzymatic activity of ASS is inhibited in a highly specific manner by the binding of a 44‐base DNA oligonucleotide carrying the ARS core consensus sequence. Other nucleic acids, rNTP and dNTP are not able to mimic the specific inhibitory effect. Single‐base substitutions in the ARS core sequence lead to a tenfold reduction in inhibition. The inhibition data corroborate the earlier report on the DNA‐binding specificity of this enzyme. The homologous enzymes from Escherichia coli and Dictyostelium discoideum do not show specific binding to single‐stranded ARS sequences and their enzymatic activity is not influenced by the presence of a 44‐base DNA oligonucleotide carrying the ARS core consensus sequence. Treatment of ASS with alkaline phosphatase leads to a loss of DNA binding and to a loss of the inhibition by DNA of the enzymatic activity which suggests that the DNA‐binding activity but not the enzymatic activity may be regulated by the phosphorylation status of the protein.

Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques. The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms. The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.