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Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail
- Gavriljuk, Konstantin, Gazdag, Emerich-Mihai, Itzen, Aymelt, Kötting, Carsten, Goody, Roger S., Gerwert, Klaus
- Proceedings of the National Academy of Sciences of the United States of America 2012 v.109 no.52 pp. 21348-21353
- Fourier transform infrared spectroscopy, GTPase-activating proteins, X-radiation, active sites, catalytic activity, glutamine, guanosine triphosphate, guanosinetriphosphatase, humans, hydrolysis, phosphates
- Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis -glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis -glutamine is replaced by a glutamine from RabGAP (trans -glutamine); this differs from the RasGAP mechanism, where the cis -glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans -arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans -glutamine and trans -arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.