Purification and Characterization of Deoxyuridine Triphophosphatase from Aquifex Aeolicus Overexpressed in Escherichia Coli
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The enzyme deoxyuridine triphosphatase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and inorganic pyrophosphate, which both regulates intracellular levels of dUTP and provides the substrate for dTMP synthesis. Since dUTPase is crucial for cell viability, its inhibition could have a wide range of medical applications. In the research of dUTPase from a range of organisms, this enzyme has a high degree of specificity for dUTP over other nucleotides. The activity of the enzyme in many organisms is also increased by the addition of magnesium, and inhibited by EDT A. In light of other research on dUTPase, examination of dUTPase from A. aeolicus can be useful as a model for analysis of other dUTPases. Since the A. aeolicus is a member of one of the earliest branching families of archaebacteria, this dUTPase may be similar to others. The A. aeolicus is also thermophilic, making its enzymes very stable. In this experiment, a method of purification which produced a suitable yield of enzyme for kinetic and NMR study was determined. The A. aeolicus enzyme was overexpressed in E. coli, allowing harvest at a large scale to prepare for such a purification. The purification included a DEAE cellulose column, followed by an affi-gel blue column, and yielded 160mg. A pH-indicator assay determined the enzyme affinity (0.3 ± 0.13uM) and rate of catalysis (0.29 ± 0.038s-1). The rate of catalysis increased with an increase in magnesium concentration. Indicating that magnesium was required for catalysis, the enzyme was inhibited by EDTA. In the presence of other divalent cations, the enzyme exhibited slight inhibition in the presence of calcium and about a 30% reduction in activity in the presence of manganese.