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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 muM) 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. (C) 2002 Elsevier Science Ltd. All rights reserved.