Nucleoside diphosphate kinase from Escherichia coli.
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).
Almaula
N
N
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Lu
Q
Q
Delgado
J
J
Belkin
S
S
Inouye
M
M
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
D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D010768:Phosphoserine; D006639:Histidine; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; C005436:phosphohistidine