MECHANISTIC STUDIES OF AICAR TRANSFORMYLASE

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DESCRIPTION (Applicant's Description): The long-term objectives of this proposal are to develop further 5-aminoimidazole-4-carboxamide ribotide transformylase/inosine monophosphate cyclohydrolase (ATIC) as a c h e m otherapeutic target for the treatment of cancer and to create methotrexate-resistant variants of ATIC suitable for use in hematopoietic stem c e ll support thereby expanding the use of methotrexate in high-dose chemotherapy protocols. The specific aims of this proposal are as follows: (1) to use pre-steady-state kinetic methods to define the minimal kinetic schemes for the two reactions catalyzed by ATIC; (2) to identify the regions of ATIC responsible for dimerization and substrate binding using approaches such as site-directed mutagenesis and protein footprinting; and (3) to develop a genetic screen in yeast to identify methotrexate-resistant variants of ATIC.