We propose to further characterize the purine biosynthetic complex which consists of glycineamide ribotide transformylase, trifunctional protein, AICAR transformylase (10-formyltetrahydrofolate-5-amino-1-ribosyl-4-imidazoyl carboxamide-5'-phosphate transformylase), serine transhydroxymethylase and trifunctional protein with the following experiments. 1) Kinetic coupling studies commencing with formate, ATP, H4-folate(Glu)3 or 5,10-CH2-H4-folate(Glu)3 and AICAR in the presence of trifunctional protein and AICAR transformylase to test whether formyl AICAR is synthesized; 2) synthesis of 5,11-CH-H4-homofolate(Glu)3 enzymatically employing the trifunctional protein activities; 3) culturing of an SV-28 cell line that becomes resistant to the 5,11-CH-H4-homofolate followed by isolation of the purine biosynthetic complex to determine if any of these protein(s) is present at greater concentrations than normal; and 4) synthesis of AICAR analogs modified at the carboxamide group so as to prevent cyclization to inosinic acid in order to uncouple the transformylase and inosinocase activities permitting mechanism studies.
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