This work covers biochemical characterization of GAP activation of the small GNBPs, Rap1 and RheB, which was achieved by a combination of techniques like infrared spectroscopy, mutational analysis and stopped flow kinetics. It was shown that RapGAP
catalyzed reaction involves an additional intermediate GTP state, the chemical cleavage reaction is reversible and Pi
release is rate limiting. The most important feature in GAP catalyzed reaction is a shift of negative charge towards the non-bridging oxygen of the β phosphate of GTP, which is more pronounced than Ras·RasGAP system. A carboxyamide
intermediate vibration was observed which in an independent study, turned out to be the catalytic asparagine residue of the GAP. RapGAP can down regulate mutations of position 61 and 12 of Rap and
is a better transition state mimic than AlFx, which indicates a dissociative mechanism. Also the in vitro GAP activity of tuberin, a homolog could be demonstrated.