Multistep pathways are commonly thought to involve substrate channeling, whereby the product of one enzyme is delivered to the active site of another enzyme. We propose to study this phenomenon in the mammalian purine biosynthetic pathway using mass spectrometry and a combination of biochemical and molecular genetic analysis. The biosynthetic pathway in eukaryotes has been evolutionarily streamlined by fusing the enzymes of several steps into separate domains in one protein, GARS-AIRS-GART (GART). In the process of studying folate antimetabolites, this lab identified, characterized and purified the tri-functional enzyme. GART is not the only multifunctional enzyme in eukaryotic purine anabolism, but in regards to substrate channeling, GART is perhaps the most interesting. GART performs the 2nd GARS), 3rd (GART) and 5th (AIRS) steps of purine biosynthesis. The intermediate 4th step is performed by phosphoribosylformylglycinamidine synthetase (FGAMS), a separate monofunctional protein. We have recently isolated the cDNA of FGAMS from a mouse cell line. Here, we propose to purify FGAMS for kinetic studies on substrate channeling in vitro and to investigate protein/protein interactions in vivo. In order to examine substrate channeling, the main kinetic studies with FGAMS will involve radioisotope dilution studies performed in the presence of tri-functional GART. Product will be assessed for specific activity and the results will determine whether intermediate products have access to solvent and therefore whether channeling is a tight or leaky process. The hypothesis that the enzymes of purine biosynthesis exist as a large complex will be examined by expression in cell culture of tagged FGAMS and analysis by mass spectrometry of complexes formed during purine synthesis. Using the advantage of TAP (tandem affinity purification)-tagged FGAMS, we will isolate FGAMS with associated protein complexes. Following gel purification, in-gel digestion, and nano-HPLC, peptides wilt be sequenced by mass spectrometry. The results of these studies should give us clear insight into the mechanics of substrate channeling, a fundamentally important phenomenon whose exact mechanism has remained elusive.
| Tallent, Sandra M; Langston, Timothy B; Moran, Richard G et al. (2007) Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins. J Bacteriol 189:7520-4 |
