PMID:10859313

Citation	Clabecq, A, Henry, JP and Darchen, F (2000) Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP. J. Biol. Chem. 275:31786-91
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M003705200
Keywords	Aluminum Compounds/metabolism; Amino Acid Sequence; Animals; Arginine/genetics; Arginine/metabolism; Binding Sites; Calcium/pharmacology; Calmodulin/pharmacology; Catalysis/drug effects; Fluorides/metabolism; GTP-Binding Proteins/metabolism; GTPase-Activating Proteins/antagonists & inhibitors; GTPase-Activating Proteins/genetics; GTPase-Activating Proteins/metabolism; Guanine Nucleotides/metabolism; Guanosine Triphosphate/metabolism; Kinetics; Molecular Sequence Data; Mutation; Protein Binding; Recombinant Fusion Proteins/metabolism; Sequence Alignment; Substrate Specificity; Thermodynamics; rab3 GTP-Binding Proteins/genetics; rab3 GTP-Binding Proteins/metabolism; rab3A GTP-Binding Protein/chemistry; rab3A GTP-Binding Protein/metabolism; ras GTPase-Activating Proteins/metabolism
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