COLLABORATION IN BASIC SCIENCE AND ENGINEERING (COBASE)

Dr. Marvin W. Makinen hosted Dr. Beata G. Vertessy at the University of Chicago from April 24, 2000 through May 26, 2000. Dr. Vertessy is a senior researcher at the Institute of Enzymology at the Hungarian Academy of Sciences.

Their research project, "Catalytically Competent Active Site Structure of dUTP-Pyrophosphatase Determined by Electron Nuclear Double Resonance," enhances understanding of metal ion specificity of E. coli dUTPase. This knowledge can be used to define important constraints about accommodation of metal ions into the active site. Molecular modeling of the enzyme structure can be carried out to account for spectroscopically defined interactions of the metal ion with inhibitors and active site residues. The preliminary work done on this project will hopefully lead to improved drug design, particularly against the enzymes of the Herpes simplex virus.

The doctors sought to further these objectives by accomplishing the following two goals: First, the collaborators planned to search for the most chemically suitable paramagnetic substitute metal cation to replace Mg2+ as the functional, natural cofactor underlying the catalytic action of dUTP-pyrophosphatase, anticipating that this would be fulfilled by either BO2+, Mn2+, or Gd3+ as spectroscopic probes of enzyme action. The second goal was to determine the stoichiometry of the binding of the paramagnetic probe with the nucleotide substrate dUTP in solution and the active site of the enzyme.

Research studies covered under the COBASE grant were divided into three areas. First, the collaborators were faced with the task of developing a method of preparation of the nucleotide dUDP. This nucleotide is no longer available through commercial sources, a fact that was not anticipated at the time of application submission. After the dUDP was synthesized, the researchers began an EPR characterization of the complex of dUDP formed with VO2+.

Finally, Drs. Makinen and Vertessy examined the suitability of the paramagnetic cations to substitute for the catalytically functional but spectroscopically silent Mg2+ cation. They found that VO2+ can function as a catalytically obligatory cofactor in dUTP hydrolysis catalyzed by dUTPase similarly to Mg2+ or Mn2+. These preliminary results indeed provide a firm basis for using VO2+ ion as a paramagnetic probe for the proposed investigation of the structural basis of metal ion : dexoyuridine nucleotide with dUTPase.

Drs. Makinen and Vertessy are currently preparing a proposal to study this topic further. They plan to submit this proposal to either the National Science Foundation or the National Institutes of Health.