Jenkins, Tracy; (1997) The kinetic mechanism of the interaction of p21ras with neurofibromin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This project involved examining the mechanism of interaction of the 21kDa guanine nucleotide binding protein, N-ras, with the catalytic domain of the GTPase-activating protein, neurofibromin. Neurofibromin was expressed as a glutathione-S-transferase (GST) fusion protein and was either cleaved with thrombin to yield neurofibromin or eluted as neurofibromin. GST fusion protein, while ras was expressed untagged. As some evidence existed as to ras and GST being dimers, the oligomeric states of the proteins involved in this study (ras, neurofibromin, GST and neurofibromin. GST) were examined initially using analytical gel filtration and later by equilibrium sedimentation analytical sedimentation. Ras and neurofibromin were found to be monomeric while neurofibromin.GST was found to be dimeric. Using a fluorescent analogue of GTP, 2'(3')-O-(N-methylanthraniloyl) GTP (mantGTP) the interaction of ras with neurofibromin was studied by stopped-flow and quenched flow techniques. The binding of the mutant ras protein, Leu6lras.mantGTP, with neurofibromin is proposed to occur as a two-step process, having an initial rapid equilibrium process with a KD of 5.2μM at 30°C in the presence of 20mM Tris.HC1 pH 7.5, 1mM MgC12 and 1mM DTT, followed by an isomerisation of the Leu61ras.mantGTP.neurofibromin complex having a rate constant of 392s-1. The binding of wild-type ras.mantGTP with neurofibromin was also shown to be a two-step process, with an initial rapid equilibrium process with a KD of 1.2μM at 30°C in the presence of 20mM Tris.HC1 pH 7.5, 1mM MgCl2 and 1mM DTT, followed by an isomerisation of the ras.mantGTP.neurofibromin complex having a rate constant of 236s-1. The isomerisation of the ras.mantGTP.neurofibromin complex is followed by cleavage of the ras-bound mantGTP with a rate constant of 26.4s-1. This cleavage process is followed by dissociation of neurofibromin from the ras.mantGDP.Pi.neurofibromin complex with a rate constant of 8.5s-1. The effects of ionic strength and temperature on the mechanism of the interaction of ras.mantGTP and neurofibromin were studied in order to provide more information about the mechanism. The mechanism of the neurofibromin catalysed GTPase of ras is discussed in relation to the hydrolysis mechanism of other nucleoside triphosphatases.

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