Single Molecule Processivity of SOS-cat, the Catalytic Core of the Ras Gef ‘son of Sevenless’

Abstract

The Ras GEF Son of Sevenless (SOS) activates the membrane-anchored G-protein Ras by catalyzing the replacement of Ras bound GDP with GTP. We have previously shown that in addition to the catalytic site, SOS has a catalytically inactive allosteric binding site for Ras, which allows SOS to localize and up-concentrate at Ras presenting membranes, dramatically increasing the Ras-GDP turnover rate. Together, the catalytic and allosteric sites form the catalytic core of SOS (SOScat). In the present work we use TIRF fluorescence microscopy to show that in vitro, single SOScat enzymes can be highly processive, remaining surface bound via Ras in the allosteric site, while turning over hundreds to thousands of Ras with the catalytic site. By confining individual SOScat enzymes to micron-scale two-dimensional supported lipid bilayer ‘reaction chambers’ we can monitor arrays of hundreds to thousands of single SOScat enzymes, probing the variability in turnover rate and processivity within the enzyme ensemble. We show that only a small fraction of the SOScat enzymes are processive, and that this fraction is modulated by the nucleotide binding state of Ras, with Ras-GTP promoting a higher fraction of processive SOScat than Ras-GDP.