Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases.
Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation. We have identified the catalytic domain within the C-terminal halves of two yeast GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases. The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively. The K(m) values for the Gyp1p and Gyp7p active fragments (143 and 42 microM, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive. We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.
Albert
S
S
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Genetics, D-37070 Göttingen, Germany.
Will
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E
Gallwitz
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D
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D001120:Arginine; D020558:GTP Phosphohydrolases