title: Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail.
abstract: 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.
authors.affiliation: Department of Biophysics,  Ruhr University Bochum, 44801 Bochum,  Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Gavriljuk
authors.forename: Emerich-Mihai
authors.initials: EM
authors.lastname: Gazdag
authors.forename: Aymelt
authors.initials: A
authors.lastname: Itzen
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008322:Mammals / Q000378:metabolism*; D008958:Models, Molecular; D017550:Spectroscopy, Fourier Transform Infrared; D020761:rab1 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; C526344:TBC1D20 protein, human; D005973:Glutamine; D006160:Guanosine Triphosphate; C529587:Rab1B protein, human; D020761:rab1 GTP-Binding Proteins