Browsing by Author "Feuerstein, Juergen W."

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    Characterisation of the metal-ion - GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O-Mn superhyperfine coupling and by kinetic methods
    (1987-01) Feuerstein, Juergen W.; Kalbitzer, Hans R.; John, Jasson; Goody, Roger S.; Wittinghofer, Alfred
    Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (10(6)-10(7) M-1 s-1) and dissociation (10(-3)-10(-5) s-1) rate constants at 0 degrees C. Dramatic differences in the rate constants were not observed for the three proteins. Under non-physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t, but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7 X 10(10) M-1 for p21c to 1.3 X 10(11) M-1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O-labelled GDP showed direct coordination of a beta-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the alpha-phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotides was estimated to be greater than 10(5) M-1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the alpha-phosphate group. These results have been used to construct a model for the interactions of Mg X GDP with the active site of p21 proteins.
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    Tucker, J. et al. Expression of p21 proteins in Escherichia coli and stereochemistry of th nucleotide-binding site. EMBO J. 5, 1351-1358
    (1986-06) Tucker, J.; Sczakiel, Georg; Feuerstein, Juergen W.; John, Jasson; Goody, Roger S.; Wittinghofer, Alfred
    v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.