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.

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.

[1]  J. Engel,et al.  Exchange of ADP, ATP and 1: N6-ethenoadenosine 5'-triphosphate at G-actin. Equilibrium and kinetics. , 1979, European journal of biochemistry.

[2]  J B Gibbs,et al.  Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Parmeggiani,et al.  Modification of elongation-factor-Tu . guanine-nucleotide interaction by kirromycin. A comparison with the effect of aminoacyl-tRNA and elongation factor Ts. , 1978, European journal of biochemistry.

[4]  E. Scolnick,et al.  Biochemical properties of normal and oncogenic ras p21 , 1985 .

[5]  A. Gilman,et al.  Biochemical properties of hormone-sensitive adenylate cyclase. , 1980, Annual review of biochemistry.

[6]  Clive R. Bagshaw,et al.  The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation. , 1974, The Biochemical journal.

[7]  R. Goody,et al.  Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide‐binding site. , 1986, The EMBO journal.

[8]  J B Hurley,et al.  Homologies between signal transducing G proteins and ras gene products. , 1984, Science.

[9]  E. Scolnick,et al.  Viral Harvey ras p21 expressed in Escherichia coli purifies as a binary one-to-one complex with GDP. , 1985, The Journal of biological chemistry.

[10]  C. Der,et al.  Activation of ras genes in human tumors does not affect localization, modification, or nucleotide binding properties of p21 , 1984, Cell.

[11]  S. Srivastava,et al.  Ras p21 proteins with high or low GTPase activity can efficiently transform NIH 3T3 cells , 1986, Cell.

[12]  R. Goody,et al.  The preparation of thiophosphate analogs of GDP and their interaction with EF‐Tu , 1979, FEBS letters.

[13]  F. Eckstein,et al.  Absolute configuration of the diastereomers of adenosine 5'-O-(1-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Leberman,et al.  Homologies in the primary structure of GTP‐binding proteins: the nucleotide‐binding site of EF‐Tu and p21. , 1984, The EMBO journal.

[15]  D. Goeddel,et al.  Comparative biochemical properties of normal and activated human ras p21 protein , 1984, Nature.

[16]  F. Jurnak.  Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. , 1985, Science.

[17]  E. De Vendittis,et al.  Hydrolysis of GTP by elongation factor Tu can be induced by monovalent cations in the absence of other effectors. , 1982, The Journal of biological chemistry.

[18]  K. Arai,et al.  Studies on the polypeptide elongation factors from E. coli. V. Properties of various complexes containing EF-Tu and EF-Ts. , 1974, Journal of biochemistry.

[19]  Y. Kaziro.  The role of guanosine 5'-triphosphate in polypeptide chain elongation. , 1978, Biochimica et biophysica acta.

[20]  R. Goody,et al.  Stereochemistry of the elongation factor Tu X GTP complex. , 1983, European journal of biochemistry.

[21]  N. Gay,et al.  Homology between human bladder carcinoma oncogene product and mitochondrial ATP-synthase , 1983, Nature.

[22]  A. Levinson,et al.  Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes , 1986, Molecular and cellular biology.

[23]  K. Halliday,et al.  Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody , 1985, Molecular and cellular biology.

[24]  I. Schlichting,et al.  31P-NMR spectra of the Ha-ras p21.nucleotide complexes. , 1986, Biochemical and biophysical research communications.

[25]  B. Clark,et al.  A model for the tertiary structure of p21, the product of the ras oncogene. , 1985, Science.

[26]  H. Kalbitzer,et al.  Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex. , 1984, European journal of biochemistry.

[27]  H. Kung,et al.  Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[28]  E. Scolnick,et al.  Yeast and mammalian ras proteins have conserved biochemical properties , 1985, Nature.

[29]  A. Abragam,et al.  Electron paramagnetic resonance of transition ions , 1970 .

[30]  M. Wigler,et al.  Purification and characterization of human H-ras proteins expressed in Escherichia coli , 1985, Molecular and cellular biology.

[31]  R. Goody,et al.  The influence of metal ion complexation on 31P-NMR parameters of phosphorothioates of guanosine nucleotides , 1984 .

[32]  H. Kalbitzer,et al.  The structure of the EF-Tu . GDP . Me2+ complex. , 1982, European journal of biochemistry.

[33]  H. Kung,et al.  Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[35]  H. Kalbitzer,et al.  Structural investigations of the Mg.ATP complex at the active site of porcine adenylate kinase using phosphorothioate analogs and electron paramagnetic resonance of Mn(II) with chiral 17O-labelled ATP analogs. , 1983, European journal of biochemistry.

[36]  C. Der,et al.  Biological and biochemical properties of human ras H genes mutated at codon 61 , 1986, Cell.

[37]  G. H. Reed,et al.  Identification of the six ligands to manganese(II) in transition-state-analogue complexes of creatine kinase: oxygen-17 superhyperfine coupling from selectively labeled ligands. , 1980, Biochemistry.

[38]  E. Scolnick,et al.  Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[39]  H. Weissbach,et al.  6 – Factors Involved in the Transfer of Aminoacyl-tRNA to the Ribosome , 1977 .

[40]  J. Feramisco,et al.  The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity , 1984, Nature.

[41]  Wim G. J. Hol,et al.  Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant , 1983, Nature.