Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP.
Small G proteins of the Rab family are regulators of intracellular vesicle traffic. Their intrinsic rate of GTP hydrolysis is very low but is enhanced by specific GTPase-activating proteins (GAPs) that switch G proteins to their inactive form. We have characterized the activity of recombinant Rab3-GAP on Rab3A in solution. The K(m) and K(d) values (75 microm) indicate a low affinity of Rab3-GAP for its substrate. The affinity is higher for the transition state analog Rab3A:GDP:AlF(x) (15 microm). The k(cat) (1 s(-)(1)) is within the range of values reported for other GAPs. A mutation in the switch I region of Rab3A disrupted the interaction with Rab3-GAP. Furthermore, Rabphilin, a putative target of Rab3, inhibited the activity of Rab3-GAP on Rab3. Therefore, the Rab3-GAP-binding site involves the switch I region of Rab3 and overlaps with the Rabphilin-binding domain. Substitution of a single arginine residue (Arg-728) of Rab3-GAP disrupted its catalytic activity but not its interaction with Rab3A. We propose that Rab3-GAP, like Ras- and Rho-GAPs, stabilizes the transition state of Rab3 and provides a critical arginine residue to accelerate the GTPase reaction.
Clabecq
A
A
CNRS UPR 1929, Institut de Biologie Physico-Chimique, 13 rue P. et M. Curie, 75005 Paris, France.
Henry
J P
JP
Darchen
F
F
D017607:Aluminum Compounds / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D005459:Fluorides / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics; D020692:rab3 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020826:rab3A GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
D017607:Aluminum Compounds; D002147:Calmodulin; D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D001120:Arginine; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride