INVESTIGATION OF THE MECHANISM OF PHOSPHORIBOSYLAMINE TRANSFER FROM GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE AMIDOTRANSFERASE TO GLYCINAMIDE RIBONUCLEOTIDE SYNTHETASE

Phosphoribosylamine (PRA) is a product of glutamine phosphoribosylpyro phosphate amido-transferase (PRPP-AT) and a substrate for glycinamide ribonucleotide synthetase (GAR-syn), the first two enzymes in the de n ovo purine biosynthetic pathway. PRA has a half-life of 5 s under phys iological conditions, hydrolyzing to ribose 5-phosphate, The instabili ty of this purine precursor brings to question how the efficiency of t ransfer from one active site to the next is ensured: Is PRA transferre d by free diffusion, or is it transferred directly from one enzyme to the next through a process defined as substrate channeling? Kinetic in vestigations of reactions containing both enzymes monitoring the appea rance of the intermediate PRA and/or the product GAR were performed an d compared with the predicted kinetics assuming a free diffusion mecha nism of transfer. A significant discrepancy exists between the free di ffusion model and the experimental data when the ratios of the two enz ymes are varied. To accommodate this discrepancy, a direct transfer me chanism is proposed that is facilitated by protein-protein interaction s: Experiments to provide evidence for these stable protein-protein in teractions including gel chromatography, fluorescence spectroscopy, ch emical cross-linking, and affinity gel chromatography; however, have a ll been unsuccessful. These results suggest that the requisite channel ing interaction between PRPP-AT and GAR-syn, which is indicated by the kinetic results, must be a transient one.