Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7

Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

Crystallographic evidence for substrate-assisted GTP hydrolysis by a small GTP binding protein

GTP hydrolysis by small GTP binding proteins of the Ras superfamily is a universal reaction that controls multiple cellular regulations. Its enzymic mechanism has been the subject of long-standing debates as to the existence/identity of the general base and the electronic nature of its transition state. Here we report the high-resolution crystal structure of a small GTP binding protein, Rab11, solved in complex with GDP and Pi. Unexpectedly, a Pi oxygen and the GDP-cleaved oxygen are located less than 2.5 A apart, suggesting that they share a proton, likely in the form of a low-barrier hydrogen bond. This implies that the gamma-phosphate of GTP was protonated; hence, that GTP acts as a general base. Furthermore, this interaction should establish at, and stabilize, the transition state. Altogether, we propose a revised model for the GTPase reaction that should reconcile earlier models into a unique substrate-assisted mechanism.     

Effect of Mg(2+) on the kinetics of guanine nucleotide binding and hydrolysis by Cdc42

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cell. Mg(2+) ions play key roles in guanine nucleotide binding and in preserving the structural integrity of GTPases. We describe here the kinetics of the interaction of GTP with the Rho family small GTPase Cdc42 in the absence and presence of Mg(2+). In contrast to the cases of Ras and Rab proteins, which require Mg(2+) for the nucleotide binding and intrinsic hydrolysis of GTP, our results show that in the absence of Mg(2+), the binding affinity of GTP to Cdc42 is in the submicromolar concentration, and the Mg(2+) cofactor has only a minor effect on the Cdc42-catalyzed intrinsic hydrolysis rate of GTP. These results suggest that the intrinsic GTPase reaction mechanism of Cdc42 may differ significantly from that of other subfamily members of the Ras superfamily.     

QM/MM modeling the Ras-GAP catalyzed hydrolysis of guanosine triphosphate

The mechanism of the hydrolysis reaction of guanosine triphosphate (GTP) by the protein complex Ras-GAP (p21(ras) - p120(GAP)) has been modeled by the quantum mechanical-molecular mechanical (QM/MM) and ab initio quantum calculations. Initial geometry configurations have been prompted by atomic coordinates of a structural analog (PDBID:1WQ1). It is shown that the minimum energy reaction path is consistent with an assumption of two-step chemical transformations. At the first stage, a unified motion of Arg789 of GAP, Gln61, Thr35 of Ras, and the lytic water molecule results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low-barrier transition state TS1. At the second stage, Gln61 abstracts and releases protons within the subsystem including Gln61, the lytic water molecule and the gamma-phosphate group of GTP through the corresponding transition state TS2. Direct quantum calculations show that, in this particular environment, the reaction GTP + H(2)O --> GDP + H(2)PO(4) (-) can proceed with reasonable activation barriers of less than 15 kcal/mol at every stage. This conclusion leads to a better understanding of the anticatalytic effect of cancer-causing mutations of Ras, which has been debated in recent years.     

Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.     

Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg(2+)GDP reveals two new binding conformations-in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the gamma-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP-bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.     

The mechanism of GTP hydrolysis by Ras probed by Fourier transform infrared spectroscopy

Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of (18)O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-(18)O(2)]GTP, NPE-[beta-(18)O(4)]GTP, or NPE-[gamma-(18)O(3)]GTP upon photolysis or during hydrolysis afforded a substantially improved mode assignment of phosphoryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hydroxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn(2+), Ras(Pro(12)), Ras(Ala(12)), and Ras(Val(12)), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP beta-PO(3)(2-) moiety by Ras mutants with higher activity, suggesting that the transition state is largely GDP-like. Analysis of the photolysis and hydrolysis FTIR spectra of the [beta-nonbridge-(18)O(2), alphabeta-bridge-(18)O]GTP isotopomer allowed us to probe for positional isotope exchange. Such a reaction might signal the existence of metaphosphate as a discrete intermediate, a key species for a dissociative mechanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a considerable amount of dissociative character. This work demonstrates that time-resolved FTIR is highly suitable for monitoring positional isotope exchange and advantageous in many aspects over previously used methods, such as (31)P NMR and mass spectrometry.     

The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

A novel magnesium-dependent mechanism for the activation of transducin by fluoride

Activation of transducin-GDP by NaF is mainly mediated by aluminofluorde or beryllofluoride complexes acting as GTP gamma-phosphate analogs. In millimolar magnesium, NaF at concentrations above 3 mM is active even in the absence of aluminium or beryllium. This activation has a Hill coefficient of 3 with respect to F-, and its rate is linear with respect to Mg2+ concentrations above 2 mM. Upon fluoride dilution, inactivation rate is hundreds of times faster than for aluminofluoride-activated T alpha GDP. We propose that at high NaF concentrations, 3 hydrogen-bonded fluorides in the gamma-phosphate site of T alpha GDP entrap a magnesium counterion and this induces the transconformation to the T alpha GTP form.     

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.     

Ras catalyzes GTP hydrolysis by shifting negative charges from gamma- to beta-phosphate as revealed by time-resolved FTIR difference spectroscopy

FTIR difference spectroscopy has been used to determine the molecular GTPase mechanism of the small GTP binding protein Ras at the atomic level. The reaction was initiated by the photolysis of caged GTP bound to Ras. The addition of catalytic amounts of the GTPase activating protein (GAP) reduces the measuring time by 2 orders of magnitude but has no influence on the spectra as compared to the intrinsic reaction. The reduced measuring time improves the quality of the data significantly as compared to previously published data [Cepus, V., Scheidig, A., Goody, R. S., and Gerwert, K. (1998) Biochemistry 37, 10263-10271]. The phosphate vibrations are assigned using 18O-labeled caged GTP. In general, there is excellent agreement with the results of Cepus et al., except in the nu(a)(alpha-PO2-) vibration assignments. The assignments reveal that binding of GTP to Ras induces vibrational uncoupling into mainly individual vibrations of the alpha-, beta-, and gamma-phosphate groups. In contrast, for unbound GTP, the phosphate vibrations are highly coupled and the corresponding absorption bands are broader. This result indicates that binding to Ras forces the flexible GTP molecule into a strained conformation and induces a specific charge distribution different from that in the unbound case. The binding causes an unusual frequency downshift of the GTP beta-PO2- phosphate vibration, whereas the alpha-PO2- and gamma-PO3(2-) phosphate vibrations shift to higher wavenumbers. The frequency downshift indicates a lowering of the bond order of the nonbridged P-O bonds of the beta-phosphate group of GTP and GDP. The bond order changes can be explained by a shift of negative charges from the gamma- to the beta-oxygens. Thereby, the GTP charge distribution becomes more like that in GDP. The charge shift appears to be a key factor contributing to catalysis by Ras in addition to the correct positioning of the attacking water. Ras appears to increase the negative charge at the pro-R beta-oxygen mainly by interaction of Mg(2+) and at the pro-S beta-oxygen mainly by interactions of the backbone NHs of Lys 16, Gly 15, and Val 14. The correct positioning of the backbone NHs of Lys 16, Gly 15, and Val 14, and especially the Lys 16 side chain, of the structural highly conserved phosphate binding loop relative to beta-phosphate therefore seems to be important for the catalysis provided by Ras.     

Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction.

The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (thioGTP) has been investigated by rapid-reaction techniques. The myosin was isolated from rabbit skeletal muscle. The steady-state intermediate of these reactions consists pre-dominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component. The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism. The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding. The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 degrees C. This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism. GDP is hydrolyzed in the presence of subfragment 1 to GMP and Pi. The subfragment 1 GTPase mechanism has a nuber if features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli.     

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.     

Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.     

Interactions of GTP with the ATP-grasp domain of GTP-specific succinyl-CoA synthetase

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20beta) and with backbone atoms provide the specificity. The gamma-phosphate interacts with the side chain of an arginine residue (Arg-54beta) and with backbone amide nitrogen atoms, leading to tight interactions between the gamma-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the gamma-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the alpha- and beta-phosphates allow the gamma-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the beta-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.     

The interactions of cell division protein FtsZ with guanine nucleotides

Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.