Tyr39 of ran preserves the Ran.GTP gradient by inhibiting GTP hydrolysis.
Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound "on" and a GDP-bound "off" state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran.GTP within the nucleoplasm and excess Ran.GDP within the cytoplasm, the hydrolysis of Ran.GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran.GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized.
Brucker
Sven
S
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Gerwert
Klaus
K
Kötting
Carsten
C
D000327:Adsorption; D000818:Animals; D020134:Catalytic Domain; D005057:Eukaryotic Cells / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D011487:Protein Conformation; D021381:Protein Transport; D014443:Tyrosine / Q000737:chemistry / Q000502:physiology*; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
D020690:GTPase-Activating Proteins; D014443:Tyrosine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020931:ran GTP-Binding Protein