(18)O Kinetic isotope effects in G protein GTPases

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DESCRIPTION (provided by applicant): The goal of the proposed research is to define the transition state and mechanism for G protein-catalyzed GTP hydrolysis. G proteins, which include members of the Ras superfamily and the alpha subunits of heterotrimeric G proteins (Galpha), are signal transducers when bound to GTP, but are inactivated by their intrinsic or GTPase activity. In vivo, GTPase activity can be accelerated by GTPase Activating Proteins. Intrinsic GTPase activity is slow and conformational changes in the catalytic site may be rate-limiting. GAP proteins accelerate intrinsic GTPase rates both by reducing the kinetic barrier to conformational changes in the G protein catalytic site and by facilitating chemical steps in the reaction. GAPs for the small G proteins Ras and Ran, and for the heterotrimeric Galpha proteins, appear to accelerate the GTPase activity of their G protein substrates by different mechanisms. Of particular interest is whether GAP proteins change the transition state for G protein-catalyzed GTPase reactions. It is proposed to measure the Kinetic Isotope Effect (KIE) for hydrolysis of specifically (18)O-labeled GTP substrates in order to determine whether conformational or chemical steps are rate-limiting for G protein and GAP-facilitated GTP hydrolysis. Primary and secondary isotope effects will be measured to determine the nature of the transition states for GTP hydrolysis catalyzed by Ras, Ran and Galpha-i1, in the presence of appropriate nucleotide exchange factors and GAPs. KIEs for GTPase reactions catalyzed by Galpha-i1 containing active site mutations shall be conducted in order to probe the role of catalytic residues in transition-state formation. A sensitive Chemical Reaction Interface coupled to an Isotope Ratio Mass Spectrometer shall be used to determine precisely the relative isotopic substitution of remotely (13)C-labeled (18)O-GTP substrates and products. The proposed methodology requires only nanomolar quantities of isotopically labeled substrates and is therefore applicable to mechanistic sudies of variety of ATPases and GTPases of significant biochemical and physiological interest.