Investigation of the Mechanism of Phosphoribosylamine Transfer from Glutamine Phosphoribosylpyrophosphate Amidotransferase to Glycinamide Ribonucleotide Synthetase

Abstract

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.

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

“…First, the transit time from one active site to the next is reduced; therefore, unfavorable equilibria, competing enzymatic transformations, or unwanted side reactions can thus be circumvented (Ushiroyama et al, 1992;Herrmann, 1995). Chemically labile intermediates can be protected from breakdown by the aqueous external environment during the transfer between distant active sites (Rudolph and Stubbe, 1995). Sequestration also provides a means of preventing a potentially toxic intermediate product from being released to the solvent (Manjasetty et al, 2003).…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…Passage of an intermediate directly from one active site to another without equilibration with the solvent is called channeling (26)(27)(28)(29)(30). Examples of reactive intermediates that are channeled from one active site to another include carbamate (31), ammonia (32), indole (33), aspartyl phosphate (34), glutamyl phosphate (35), and phosphoribosylamine (26).…”

“…The PcpB:PcpD system is not the first case in which kinetic evidence for a protein-protein interaction is compelling, but a physical interaction cannot be detected. Other examples include aspartyl kinase: aspartate semialdehyde dehydrogenase (34), glutamyl kinase: glutamate semialdehyde dehydrogenase (35), and glutamine phosphoribosylpyrophosphate amidotransferase:glycinamide ribonucleotide synthetase (26). The hypothesis that a proteinprotein interaction between PcpD and PcpB is responsible for the sequestration of TCBQ is supported by experiments showing that TCBQ is released to the solvent when PcpD-FeS is present, even though this truncated enzyme is kinetically competent for reduction of TCBQ.…”

“…Such interactions are often not amenable to detection by methods such as native gel electrophoresis, gel filtration, chemical cross-linking, or surface plasmon resonance (26,40,41) although their importance is well recognized (42). A transient interaction between PcpB and PcpD might be expected given that PcpB is expected to undergo The rates of reaction of TCBQ with β−ME and PcpD under the experimental conditions.…”

Purification, crystallization and preliminary X-ray diffraction data from selenomethionine glycinamide ribonucleotide synthetase

“…A recent kinetic study (Rudolph & Stubbe, 1995) suggested that the transfer of this unstable intermediate from PRPP-AT to GAR-syn takes place by a process de®ned as substrate channeling, which would be facilitated by speci®c protein±protein interactions. The X-ray structure of PRPP-AT has been previously determined (Kim et al, 1996).…”