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Lascu, Ioan; Gonin, Philippe

doi:10.1023/a:1005532912212

Cited by 173 publications

(81 citation statements)

References 61 publications

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“…We could model these proton release events with Michaelis-Menten kinetics in which a rapid equilibrium (k 1 , k Ϫ1 ) precedes the rate-limiting step (k 2 ϭ k cat ). Global fits to the single and multiple turnover time courses using the k 1 , k Ϫ1 , and k 2 floating parameters of Scheme (26,30). We took advantage of the fact that the phosphorylated enzyme intermediate of the NDPK reaction is long lived in the absence of Mg 2ϩ and thus can be separated from the phosphate donor nucleotides (26).…”

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confidence: 99%

Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

Tóth

Varga

Kovács

et al. 2007

Journal of Biological Chemistry

139

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Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzymeproduct complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize ␥-32 P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.dUTPase is the unique enzyme that specifically hydrolyzes the ␣-␤ pyrophosphate bond of dUTP to yield dUMP and PP i(1). The enzyme is essential in maintaining DNA integrity in dividing cells (2, 3). Its activity is responsible for setting the physiological dUTP/dTTP concentration ratios (1:24) (4), thus preventing high rates of uracil incorporation into newly synthesized DNA. Although uracil in DNA is tolerated to a certain level by the base excision DNA repair mechanisms, higher levels of uracil in DNA trigger double-strand breaks and lead to cell death (5). Several lines of evidence show that up-regulated dUTPase is responsible for desensitizing tumors to drugs inhibiting the thymidylate synthase pathway, thus acting as an important survival factor for tumor cells (6, 7). Increased levels of the nuclear isoform of the enzyme correlate to worsened prognosis of several tumors, as revealed by detailed analysis of tissue samples (8, 9). dUTPase has therefore emerged as a high potential anticancer drug target, which possesses several additional, possibly advantageous features for drug design. Unlike most nucleotide-metabolizing enzymes, dUTPase is extremely specific to its substrate nucleotide, potenti...

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confidence: 99%

Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

Tóth

Varga

Kovács

et al. 2007

Journal of Biological Chemistry

139

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“…Succinyl-CoA ligase converts succinyl-CoA to succinate with the generation of GTP in the tricarboxylic acid cycle (5). Nucleoside diphosphate kinases catalyze the transfer of a ␥ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (6). The high energy phosphate is usually supplied by ATP, and the enzyme regulates the crucial balance between ATP and GTP or other NTPs.…”

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confidence: 99%

GTP Is Required for Iron-Sulfur Cluster Biogenesis in Mitochondria

Amutha

Gordon

et al. 2008

Journal of Biological Chemistry

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Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

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“…NDPKs were originally identified as essential housekeeping enzymes required for the synthesis of nucleoside triphosphates by catalyzing the transfer of the ␥-phosphoryl groups from nucleoside triphosphates to nucleoside diphosphates via a phosphohistidine 118 enzyme intermediate, and they play a role in maintaining intracellular nucleotide concentrations (10). Altered expression of NDPK is also reportedly involved in many cellular processes, including oncogenesis (11,12), cellular proliferation (13), differentiation (14,15), motility (16), development (17), DNA repair (18), and apoptosis (19).…”

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confidence: 99%