title: Nucleoside diphosphate kinase from Escherichia coli.
abstract: Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: N
authors.initials: N
authors.lastname: Almaula
authors.forename: Q
authors.initials: Q
authors.lastname: Lu
authors.forename: J
authors.initials: J
authors.lastname: Delgado
authors.forename: S
authors.initials: S
authors.lastname: Belkin
authors.forename: M
authors.initials: M
authors.lastname: Inouye
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D017421:Sequence Analysis
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D010768:Phosphoserine; D006639:Histidine; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; C005436:phosphohistidine