Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage.
Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.
Godson
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Biochemistry Department, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA. godsog01@mcrcr6.med.nyu.edu
Schoenich
J
J
Sun
W
W
Mustaev
A A
AA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol; D004926:Escherichia coli; D007501:Iron / Q000737:chemistry* / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D014357:Trypsin
D002413:Cations, Divalent; D010761:Phosphorus Radioisotopes; D007501:Iron; D019915:DNA Primase; D014357:Trypsin; D008274:Magnesium; D004229:Dithiothreitol