Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions

J Mol Biol. 2002 Dec 6;324(4):763-74. doi: 10.1016/s0022-2836(02)01136-1.

Abstract

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.

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Enzyme Activation
  • Escherichia coli / metabolism
  • Fluorescent Dyes*
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Proteins / metabolism*
  • GTPase-Activating Proteins / metabolism*
  • Guanosine Diphosphate / metabolism
  • Manganese / chemistry
  • Molecular Conformation
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence
  • p120 GTPase Activating Protein
  • rap GTP-Binding Proteins / metabolism
  • ras GTPase-Activating Proteins / metabolism
  • ras Proteins / metabolism

Substances

  • Fluorescent Dyes
  • GTPase-Activating Proteins
  • p120 GTPase Activating Protein
  • ras GTPase-Activating Proteins
  • Guanosine Diphosphate
  • Manganese
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • RAP1B protein, human
  • Proto-Oncogene Proteins p21(ras)
  • rap GTP-Binding Proteins
  • ras Proteins