Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPAGene

Abstract

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC 3.6.1.19). 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.

Crystal Structure of Human Inosine Triphosphatase

“…2C). Xanthine nucleotides are also ITPA substrates (2). Additional specificity in xanthine binding may be obtained by a putative hydrogen bond between the 2-keto oxygen of the xanthine base and the Asp-152 backbone amide.…”

“…ITP, dITP, and xanthine triphosphate are converted with similar kinetics, whereas the enzyme has very low affinity for other nucleotides. The pH optimum of ITPA is ϳ10 (2,4,5).…”

“…The location of the substrate binding site has been postulated on the basis of weak binding of the non-physiological ATP analog, AMPPNP, to the Methanococcus enzyme (13). However, the mode of substrate binding proposed could not account for the strong binding preference for ITP, dITP, and xanthine triphosphate over other nucleotides (2,14 We have determined the crystal structure of human ITPA alone and in complex with one of its physiological substrates, ITP, and the products of hydrolysis, IMP ϩ PP i . Our results identify the substrate and Mg 2ϩ binding site of ITPA and show that it is different from the site previously proposed for this class of enzymes.…”

“…ITPA orthologs are found in organisms from all kingdoms. In human and mouse, the protein is ubiquitously expressed (2,3). Human ITPA is a 194-amino acid homodimer that is reliant upon Mg 2ϩ or Mn 2ϩ ions for catalytic activity.…”

ITPase-deficient mice show growth retardation and die before weaning

“…1,2 In the case of deaminated purine bases, such as hypoxanthine, it is considered that inosine triphosphate pyrophosphatase (ITPase), endonuclease V and hypoxanthine-DNA glycosylase have a role in eliminating the deaminated bases from DNA. [16][17][18][19] Saparbaev and Laval 19 reported that alkylpurine-DNA glycosylases engage in hypoxanthine-DNA glycosylase activity both in Escherichia coli and mammalian cells. The alkylpurine-DNA glycosylase-deficient (Mpg knockout) mouse has features of impaired base excision repair of alkylation-induced DNA damage, and increased sensitivity to methyl methanesulfonate and streptozotocininduced diabetes.…”