The Human Glycinamide Ribonucleotide Transformylase Domain: Purification, Characterization, and Kinetic Mechanism

Glycinamide ribonucleotide transformylase catalyzes the third reaction ofde novopurine biosynthesis, namely, the conversion of glycinamide ribonucleotide toN-formylglycinamide ribonucleotide, with concomitant conversion of 10-formyltetrahydrofolate to tetrahydrofolate. This activity has been shown to be a target for cancer chemotherapy, which has generated renewed interest in both the enzyme and the pathway. Moreover, in higher eukaryotes this activity constitutes the C-terminal domain of a monomeric protein which also catalyzes two additional reactions ofde novopurine biosynthesis. In this study, the human glycinamide ribonucleotide transformylase domain has been expressed to high levels inEscherichia coliand purified to homogeneity. Our improved expression–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 been established as ordered–sequential, with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.