Enzymatic properties and inhibition by single-stranded autonomously replicating sequences of adenylosuccinate synthase from Saccharomyces cerevisiae.
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.
Gallert
K C
KC
Lehrstuhl Biochemie, Universität Bayreuth, Germany.
Ohanjan
T
T
Daignan-Fornier
B
B
Lottspeich
F
F
Krauss
G
G
D000265:Adenylosuccinate Synthase / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; D000265:Adenylosuccinate Synthase