THE HUMAN GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE DOMAIN - PURIFICATION, CHARACTERIZATION, AND KINETIC MECHANISM

Glycinamide ribonucleotide transformylase catalyzes the third reaction of de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide to N-formylglycinamide ribonucleotide, with concomitan t conversion of 10-formyltetrahydrofolate to tetrahydrofolate. This ac tivity has been shown to be a target for cancer chemotherapy, which ha s generated renewed interest in both the enzyme and the pathway, Moreo ver, in higher eukaryotes this activity constitutes the C-terminal dom ain of a monomeric protein which also catalyzes two additional reactio ns of de novo purine biosynthesis. In this study, the human glycinamid e ribonucleotide transformylase domain has been expressed to high leve ls in Escherichia coli and purified to homogeneity, Our improved expre ssion-purification system produces the desired activity exclusively in a soluble form and in higher abundance than previously achieved. The kinetic constants have been determined and the kinetic mechanism has b een established as ordered-sequential, with the folate substrate bindi ng first, The correspondence of these data to those obtained for the g lycinamide ribonucleotide transformylase activity of the mammalian tri functional enzyme indicates that the recombinant enzyme is fully funct ional. (C) 1997 Academic Press.