Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme.
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 enzyme-product 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 gamma-(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.
Tóth
Judit
J
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, 1113 Budapest, Hungary. Electronic address: tothj@enzim.hu.
Varga
Balázs
B
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, 1113 Budapest, Hungary.
Kovács
Mihály
M
Eötvös Loránd University, Budapest, Hungary.
Málnási-Csizmadia
András
A
Eötvös Loránd University, Budapest, Hungary.
Vértessy
Beáta G
BG
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29, 1113 Budapest, Hungary. Electronic address: vertessy@enzim.hu.
D020106:Acrylamide / Q000737:chemistry; D016880:Anisotropy; D002384:Catalysis; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008968:Molecular Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
D008024:Ligands; D020033:Protein Isoforms; D012997:Solvents; D020106:Acrylamide; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; C021895:nucleoside triphosphate pyrophosphatase