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 p2lv, 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 GDPaS and GTPaS. It has low specificity for the Sp isomer of GTP,BS. Together with the data for GDP,BS and GTP-yS 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.

Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (lo6 -lo7 M-' s-') and dissociation -10-5s-') rate constants at 0°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 p21, than for the cellular form p21, or the T24 mutant p21,, 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" M-' for p21, to 1.3 x 10'' M-' for p21,. Electron paramagnetic resonance (EPR) measurements on Mn2 + bound together with stereospecifically ' 70-labelled GDP showed direct coordination of a P-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the a-phosphate oxygens at the active site of all three proteins.The association constant of Mn(I1) to p21 proteins in the absence of nucleotides was estimated to be > lo5 M-' .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 a-phosphate group. These results have been used to construct a model for the interactions of Mg . GDP with the active site of p21 proteins.A deeper insight into the role of oncogenes during cell transformation can only be obtained by understanding the function of the proteins they encode. The products of the ras oncogenes, the p21 proteins (for reviews, see [l, 21) are thought to be related to the group of guanine nucleotide (G) regulatory proteins such as transducin and elongation factor Tu (EFTu), because of significant sequence homology and similar enzymatic properties [3 -71. It is characteristic for these proteins that their regulatory function is modulated by GTP and GDP binding or, more precisely, by the binding of the corresponding Mg2+ complexes. A common property of all known G proteins is a low but clearly detectable hydrolytic activity in the absence of appropriate physiological stimulators [2, 8 -101.The GTPase activity of p21 is lowered in oncogenic forms of the protein [ l l -141. There are, however, conflicting reportes concerning the relationship of this lower GTPase activity to the transforming properties of the proteins [15-171. Surprisingly, it is generally believed that the nucleotide binding ability is unaltered in the transforming mutant proteins [2, 11,15,[17][18][19][20]. However, it is probable that there are structural differences in the nucleotide binding site between the cellular and mutant forms of p21 which are responsible for the altered GTPase activity. Obviously, knowledge of the structural properties of the nucleotide binding site is essential for the understanding of these differences an...

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF‐Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)‐(Rp)‐[β‐17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line‐broadening of the EPR spectrum of Mn2+ at the active site of EF‐Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF‐Tu · GDP · Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2′(3′)‐O‐(N‐methylanthraniloyl)‐GDP is 6.2 × 10−3s−1 at 25°C and pH 6.8], we conclude that the broadened signal arises from the EF‐Tu · Mn · GDP · Pi complex, the predominant steady‐state species. During the hydrolysis of GTP the Mn2− remains bound to the β‐phosphate oxygen of GDP which arises from the βpro‐S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF‐Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)‐[β‐17O]GTP in the corresponding complex in the presence of EF‐Ts.

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