Rate Of Nucleotide Hydrolysis Research Articles

Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration.

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.

Water Molecules in the Nucleotide Binding Cleft of Actin: Effects on Subunit Conformation and Implications for ATP Hydrolysis

In the monomeric actin crystal structure, the positions of a highly organized network of waters are clearly visible within the active site. However, the recently proposed models of filamentous actin (F-actin) did not extend to including these waters. Since the water network is important for ATP hydrolysis, information about water position is critical to understanding the increased rate of catalysis upon filament formation. Here, we show that waters in the active site are essential for intersubdomain rotational flexibility and that they organize the active-site structure. Including the crystal structure waters during simulation setup allows us to observe distinct changes in the active-site structure upon the flattening of the actin subunit, as proposed in the Oda model for F-actin. We identify changes in both protein position and water position relative to the phosphate tail that suggest a mechanism for accelerating the rate of nucleotide hydrolysis in F-actin by stabilizing charge on the β-phosphate and by facilitating deprotonation of catalytic water.

Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2'-(or -3')-O-(N-methylanthraniloyl)-beta,gamma-imidoguanosine 5'-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K(+), which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K(+) coordination, and mutation of this residue abolished K(+)-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K(+)-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics

The Ras family of small GTPases control diverse signaling pathways through a conserved "switch" mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2'(3')-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is approximately 10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.

Postictal serum nucleotidases activities in patients with epilepsy

Adenosine, a potent anticonvulsant, can be produced in the body by the hydrolysis of adenine nucleotides through the action of ecto- or soluble nucleotidases. Changes in nucleotide hydrolysis occur after pentylenetetrazol-induced epileptic events. We evaluated serum ATP, ADP and AMP hydrolysis rates and soluble nucleotide phosphodiesterase (PDEase) activity at 5, 10, 15, 30 and 60 min, and 12h following an epileptic event. Fifteen patients (seven female, eight male; mean age 15.5 years) were included in the study. The type of seizure was generalized in four patients and was localization related in the remaining 11. There were no differences in adenine nucleotide hydrolysis rates between patients and healthy subjects in the interictal stage. In comparison with controls, ATP, ADP and AMP hydrolysis rates were significantly increased at 5 min (53+/-1.4%, 79.2+/-2.8% and 37.0+/-2.6%, respectively) and up to 30 min following the epileptic event. In contrast to ADP and AMP, ATP hydrolysis remained significantly increased at 60 min (71.4+/-1.6%), returning to the basal level after 12h. Serum PDEase activity was also significantly higher in the patients than in healthy subjects, peaking at 15 min (61+/-2.9%) and remaining significantly increased up to 60 min (4.6+/-1.2%) following the epileptic episode. Globally, the variations in the postictal serum ADP hydrolysis rate almost overlapped those of AMP hydrolysis, whereas changes in the ATP hydrolysis rate overlapped those of PDEase activity. The clinical significance of this elevation in postictal soluble serum nucleotidase activity remains to be clarified. However, it is possible to hypothesize that the higher nucleotidase activity might play a role in the modulation of epileptic events.

Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1: the effects of beryllium fluoride

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the alpha- and beta-subunit homooligomers (alpha16mer or beta16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both alpha16mer and beta16mer with equal efficiency. This indicated that BeFx replaces the gamma-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin-ADP-BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin-ADP-BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of alpha16mer and beta16mer are likely similar although there were some apparent differences. While beta16mer-mediated GFP refolding in the presence of ATP-BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP-PNP, the folding mediated by the alpha16mer that proceeded with much lower yields. A mutant of alpha16mer, trapalpha, which traps the unfolded and partially folded substrate protein, did not affect the ATP-BeFx-dependent GFP folding by beta16mer but it suppressed that mediated by alpha16mer to the level of spontaneous folding. These results suggested that beta16mer differed from the alpha16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis.

Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads.

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.

Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing.

ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.

Biochemical Defects in Retina-specific Human ATP Binding Cassette Transporter Nucleotide Binding Domain 1 Mutants Associated with Macular Degeneration

The retina-specific human ABC transporter (ABCR) functions in the retinal transport system and has been implicated in several inherited visual diseases, including Stargardt disease, fundus flavimaculatus, cone-rod dystrophy, and age-related macular degeneration. We have previously described a general ribonucleotidase activity of the first nucleotide binding domain (NBD1) of human ABCR (Biswas, E. E. (2001) Biochemistry 40, 8181-8187). In this communication, we present a quantitative study analyzing the effects of certain disease-associated mutations, Gly-863 --> Ala, Pro-940 --> Arg, and Arg-943 --> Gln on the nucleotide binding, and general ribonucleotidase activities of this domain. NBD1 proteins, harboring these mutations, were created through in vitro site-specific mutagenesis and expressed in Escherichia coli. Results of the enzyme-kinetic studies indicated that these mutations altered the ATPase and CTPase activities of NBD1. The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. On the other hand, the R943Q mutation had small, but detectable reduction in its nucleotidase activity and nucleotide binding affinity. We have measured the nucleotide binding affinities of NBD1 protein and its mutants quantitatively by fluorescence anisotropy changes during protein binding to ethenoadenosine ATP (epsilonATP), a fluorescent ATP analogue. We have correlated the dissociation constant (K(D)) and the rates of nucleotide hydrolysis (V(max)) of NBD1 and its mutants with the available genetic data for these mutations.

Polymerization of FtsZ, a Bacterial Homolog of Tubulin: IS ASSEMBLY COOPERATIVE?

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis. In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or "isodesmic") assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D).

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility.

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.

Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

Nucleotide hydrolysis regulates the dynamics of actin filaments and microtubules.

Actin filaments and microtubules are major dynamic components of the cytoskeleton of eukaryotic cells. Assembly of these polymers from monomeric actin or tubulin occurs with expenditure of energy, because ATP (or GTP) tightly bound to actin (or tubulin) is irreversibly hydrolysed during polymerization. Therefore, actin filaments an microtubules are dissipative structures. Our purpose has been to understand how the dissipation of chemical energy perturbs the laws of reversible helical polymerization defined by Oosawa, and affects the dynamics of these polymers. A kinetic study has shown that nucleotide is hydrolysed on the polymer within at least two steps consecutive to the incorporation of the monomer: cleavage of the gamma-phosphoester bond followed by the slower release of Pi; only the second reaction appears reversible. Pi release, and not cleavage of the gamma-phosphate, is linked to the destabilization of protein-protein interactions in the polymer, and therefore plays the role of a conformational switch. The dynamic properties of the polymer in the NTP- and NDP-Pi intermediate states of the assembly process have been investigated using non-hydrolysable analogues of nucleotides and structural analogues of Pi, AlF4- and (BeF3-, H2O). Because nucleotide hydrolysis is uncoupled from polymerization, actin filaments and microtubules grow with a 'cap' of terminal NTP- and NDP-Pi-subunits that interact strongly, and prevent the rapid depolymerization of the unstable core of the polymer formed of NDP-subunits. The fact that the dynamic properties of the polymer are affected by bound nucleotide results in a nonlinear dependence of the rate of elongation on monomer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide and bivalent cation specificity of the insulin-granule proton translocase.

1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from adenohypophyseal tissue, adrenal chromaffin granules and yeast vacuoles. The insulin-granule activity thus appears to be a type of proton translocase, which is characteristic of intracellular storage vesicles in eukaryotic cells.

PH-induced changes in the reactions controlled by the low- and high-affinity Ca2+-binding sites in sarcoplasmic reticulum.

The effect of pH on the Ca2+-binding sites of high and low affinity, located respectively on the outer and inner surfaces of the sarcoplasmic reticulum membrane, was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumualted by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the two Ca2+-binding sites could be controlled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, we observed that the affinity of both the high- and low-affinity sites increased three to four orders of magnitude when the pH of the assay medium was increased from 6.1 to 8.65.