SMGGDS STABILIZES NUCLEOTIDE-BOUND AND NUCLEOTIDE-FREE FORMS OF THE RAC1 GTP-BINDING PROTEIN AND STIMULATES GTP GDP EXCHANGE THROUGH A SUBSTITUTED ENZYME MECHANISM/

The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associat ed with membrane ruffling. These functions are controlled by the GTP-b ound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5'-[gamma- thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and C DC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutel y required for the interaction with smgGDS. Interestingly, the activit y of smgGDS toward Rad could not be observed in a [H-3]GDP/GTP exchang e assay under conditions where it stimulated incorporation of GTP[gamm a S] into Rad. We determined that smgGDS prevented the loss of Rac1 ac tivity during the [H-3]GDP/GTP exchange assay by demonstrating the abi lity of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly coun terbalanced by the ability of smgGDS to stimulate the release of [H-3] GDP from Rac1, thereby producing no net observable effect in the excha nge assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rad in a nucleotide- free form after release of GDP, indicating that the reaction between R ac1 and smgGDS involves a substituted enzyme mechanism.