Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions.
Downregulation of small guanine nucleotide-binding proteins (GNBPs) requires the interaction with their corresponding GTPase-activating proteins (GAPs), which increase the slow intrinsic GTPase reaction by several orders of magnitude. On the basis of the structure of H-Ras in complex with the catalytic domain of p120-GAP, we have developed a set of site-specifically labelled Ras-variants, one of which turned out to be particularly sensitive for studying the interaction with Ras-specific GAPs. This specific fluorescent reporter group and the use of manganese to increase the rate of the chemical reaction step allowed us to identify differences in the rate-limiting step of either the GAP-334 or NF1-333 catalyzed reaction. The assay was also applied to study the interaction of the Ras-related protein Rap1B with Rap1GAP, for which no detailed kinetic analysis was available. Single-turnover experiments of this reaction show that the low affinity of the complex (50 microM) is due to a slow association rate as well as a fast dissociation rate. RapGAP promotes AlFx binding to Rap1B, even though it does not contain a catalytic arginine. The rate-limiting step of the RapGAP catalysed reaction is release of inorganic phosphate, which is about five times slower than the chemical cleavage step. Our data reveal marked differences in GAP/target interactions even between closely related systems and suggest that the fluorescent reporter group method might be generally applicable to many other GNBPs and their cognate GAPs.
Kraemer
Astrid
A
Max-Planck-Institut für molekulare Physiologie, Abt. Physikalische Biochemie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Brinkmann
Thilo
T
Plettner
Ina
I
Goody
Roger
R
Wittinghofer
Alfred
A
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D020729:p120 GTPase Activating Protein; D020668:rap GTP-Binding Proteins / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; D020729:p120 GTPase Activating Protein; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D008345:Manganese; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C116753:RAP1B protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins; D018631:ras Proteins