STRUCTURAL STUDIES OF ANTITUMOR AMIDOHYDROLASES

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Asparaginase therapy selectively inhibits certain cancers by starving the tumor cells of a required nutrient - asparagine. The E. coli enzyme is used in the treatment of acute lymphoblastic leukemia. Several glutaminase-asparaginases are active against asparaginase-resistant tumors, and an enzyme from Pseudomonas 7A has been shown to be active against a variety of ascites and solid tumors in mice. We propose to use X-ray diffraction techniques to determine the crystal structures of the Pseudomonas 7A and Acinetobacter glutaminasificans glutaminase-asparaginases, and of a Vibrio succinogenes asparaginase, at resolutions of 2-2.5 Omicron. The enzymes are tetramers with molecular weights of about 140,000 daltons. A 6 Omicron resolution map of the Acinetobacter enzyme has been obtained utilizing form III crystals, which contain one subunit per asymmetric unit. Patterson rotation methods have been used to determine the symmetry and orientation of the Pseudomonas tetramer. The synthesis of several heavy atom containing compounds for specific labeling of the glutaminase-asparaginases is proposed, which will facilitate the use of multiple isomorphous replacement and anomalous scattering methods. Molecular replacement techniques also will be used, especially for the Vibrio asparaginase. The enzyme structures will be compared to obtain a detailed picture of the catalytic mechanism and structure of the active sites, and to determine the relationships between molecular structure and antitumor activity. In addition, preliminary crystallographic studies of a novel indolyl-3-alkane Alpha-hydroxylase from Pseudomonas XA will be carried out.