Kinetic and Structural Analysis of the Mg2+ -binding Site of the Guanine Nucleotide-binding Protein p21 H-ras.

The coordination and binding of the Mg2+ ion in the nucleotide−binding site of p21 have been investigated using site−directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30− or 16−fold on substituting Ser−17 or Asp−57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr−35 is only complexed to Mg2+ in the GTP conformation and both Asp−57 and Ser−17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine−5'−(beta,gamma−imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide− binding site compared to wild−type protein. The mutant proteins show reduced GTPase rates, which are not stimulated by the GTPase−activating protein GAP. p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21−induced survival of primary neuron

Biomimetische Metalloxo-Modellkomplexe: Kinetiken, Redoxpotentiale und mechanistische Studien

Die vorliegende Arbeit befasst sich mit der Untersuchung hochvalenter Metalloxo-Komplexe. Insbesondere werden die Reaktivitäten und der Einfluss elektronischer Eigenschaften auf diese untersucht. Dabei liegt das Hauptaugenmerk darauf, eine Korrelation zwischen diesen beiden Eigenschaften zu ermitteln, damit umgekehrt Rückschlüsse auf die Reaktivität von Verbindungen mit bekannten elektronischen Eigenschaften gezogen werden können. Des Weiteren werden die Komplexe von Ligandensystemen mit identischem Rückgrat und unterschiedlichen elektronischen Eigenschaften des Donorsets miteinander verglichen, um den Einfluss unterschiedlicher Substitutionsmuster am Liganden auf die Reaktivität zu erkennen. Der erste Teil dieser Arbeit geht auf die Kinetiken der Oxidationsreaktionen mit Bispidineisen(IV)oxo-Komplexen ein. Hierbei wird auf die drei verschiedenen Reaktionstypen, CH-Aktivierung von Alkanen, Epoxidierung von Alkenen und Sauerstoff-Übertragung eingegangen. Ebenso wird die Abhängigkeit der Reaktionsgeschwindigkeit von der Protonenkonzentration untersucht. Im zweiten Teil dieser Arbeit werden die Redoxpotentiale behandelt. Für die Metalloxo-Komplexe wird der reine, nicht-Protonengekoppelte Elektronenübergang spektro-photometrisch untersucht und somit das M(n/n-1)+-Reduktionspotential bestimmt. Auch hier wird der Einfluss bei Zugabe von Protonen auf die Oxidation von Ferrocen gezeigt. Der dritte und letzte Teil dieser Arbeit befasst sich mit der Bestimmung der Reorganisationsenergie des Elektronentransfers von Eisen(IV) zu Eisen(III) in den Bispidinkomplexen und erörtert den Einfluss dieser Größe auf die Reaktions-geschwindigkeits¬konstanten der Oxidations- und Oxygenierungsreaktionen

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity

The small G protein MglA and its cognate GAP MglB exemplify a new type of GTPase activation mechanism. In contrast to other Ras-like proteins, the key 'arginine finger' is provided not by the GAP, but by MglA itself

STUDIES ON THE STRUCTURE AND MECHANISM OF H-RAS P21

Current knowledge of the structure of H-ras p21 is reviewed with particular emphasis on the interaction between guanine nucleotides and the active site of the protein. The nature of the conformational change induced by GTP hydrolysis is discussed. The major change is seen in the region known as the effector loop (loop 2), with significant but less well-defined changes occurring in loop 4, which is implicated in the GTPase reaction. Other evidence concerning the mechanism of GTP hydrolysis and its activation by GAP (GTPase-activating protein) is also discussed. Evidence regarding the rate limiting step in the p21 GTPase reaction, and the manner in which this and possibly other steps are accelerated by GAP, is inconclusive

Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.

The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS

Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides

The interaction of the protein product of the H-ras oncogene with a series of nucleoside di- and triphosphates has been examined to investigate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in a simple filter binding assay to give semiquantitave estimates of their affinities. For more weakly binding nucleotides or to obtain quantitative data, a transient kinetic method was used which was based on determination of the association and dissociation rate constants. The results obtained indicate that substantial modification of the sugar or phosphate structure is tolerated with little or moderate loss of affinity, but that large losses in affinity occur on modification of the base structure. In particular, replacing the guanine by an adenine residue leads to a dramatic loss of affinity. Thus, discrimination against ATP and ADP is very high (relative affinities of ATP and GTP 1:10(7)). This is due not only to loss of positive (stabilizing) interactions, but especially to the introduction of negative ones