Investigation of the mechanism of phosphoribosylamine transfer from glutamine phosphoribosylpyrophosphate amidotransferase to glycinamide ribonucleotide synthetase.
Phosphoribosylamine (PRA) is a product of glutamine phosphoribosylpyrophosphate amidotransferase (PRPP-AT) and a substrate for glycinamide ribonucleotide synthetase (GAR-syn), the first two enzymes in the de novo purine biosynthetic pathway. PRA has a half-life of 5 s under physiological conditions, hydrolyzing to ribose 5-phosphate. The instability of this purine precursor brings to question how the efficiency of transfer from one active site to the next is ensured: Is PRA transferred by free diffusion, or is it transferred directly from one enzyme to the next through a process defined as substrate channeling? Kinetic investigations of reactions containing both enzymes monitoring the appearance of the intermediate PRA and/or the product GAR were performed and compared with the predicted kinetics assuming a free diffusion mechanism of transfer. A significant discrepancy exists between the free diffusion model and the experimental data when the ratios of the two enzymes are varied. To accommodate this discrepancy, a direct transfer mechanism is proposed that is facilitated by protein-protein interactions. Experiments to provide evidence for these stable protein-protein interactions including gel chromatography, fluorescence spectroscopy, chemical cross-linking, and affinity gel chromatography; however, have all been unsuccessful. These results suggest that the requisite channeling interaction between PRPP-AT and GAR-syn, which is indicated by the kinetic results, must be a transient one.
Rudolph
J
J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Stubbe
J
J
D000582:Amidophosphoribosyltransferase / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases; D004058:Diffusion; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism*; D011485:Protein Binding; D012267:Ribosemonophosphates / Q000378:metabolism*
D012267:Ribosemonophosphates; C008891:phosphoribosylamine; D010754:Phosphoribosyl Pyrophosphate; D000582:Amidophosphoribosyltransferase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022387:phosphoribosylamine-glycine ligase