3-D STRUCTURES OF URIDINE & THYMIDINE PHOSPHORYLASES

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The objective is to determine the three-dimensional structures of uridine phosphorylase and thymidine phosphorylase. The pyrimidine phosphorylases may be responsible for the degradation of certain nucleoside analogs of potential chemotherapeutic value. It has been suggested that pyrimidine phosphorylase inhibitors might be useful as chemotherapeutic agents either by enhancing the efficacy of pyrimidine nucleoside analogs or by interfering with pyrimidine base salvage. In addition, the pyrimidine phosphorylases have been utilized in the enzymatic approach to laboratory synthesis of nucleoside analogs. The ultimate goal is to provide precise structural data which can be used in two possible ways. One way is to guide efforts to synthesize new inhibitors of the mammalian enzymes. The second way is to suggest structural modifications which might alter the specificity and stability of the E. coli enzyme leading to a more useful synthetic tool. Such modifications could be performed using standard techniques of genetic engineering. Single-crystal X-ray diffraction methods will be used to determine the crystal structures of uridine phosphorylase and thymidine phosphorylase. Crystals of both enzymes from E. coli have been grown and are suitable for high-resolution crystallagraphic analysis. The structures will be determined using multipe-isomorphous- replacement techniques and refined using intensity data measured with oscillation photography and synchrotron radiation. Once the structures are known, the active sites will be characterized by examining substrate, substrate analog and inhibitor complexes of the enzymes. It is hoped that the high-resolution structures of uridine phosphorylase and thymidine phosphorylase will provide important information towards the understanding of these key enzymes.