Cloning, expression, and characterization of a human inosine triphosphate pyrophosphatase encoded by the itpa gene.
ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, People's Republic of China.
S
S
Lin
A G
AG
McLennan
K
K
Ying
Z
Z
Wang
S
S
Gu
H
H
Jin
C
C
Wu
W
W
Liu
Y
Y
Yuan
R
R
Tang
Y
Y
Xie
Y
Y
Mao
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002850:Chromatography, Gel; D002890:Chromosomes, Human, Pair 20; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D016208:Databases, Factual; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007293:Inosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D021101:Radiation Hybrid Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014158:Transcription, Genetic; D000097630:Inosine Triphosphatase
D018076:DNA, Complementary; D007293:Inosine Triphosphate; D011755:Pyrophosphatases; D012313:RNA; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000097630:Inosine Triphosphatase; C065326:xanthosine 5'-triphosphate