Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.     

Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl-phosphate synthetase I. Studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine.

The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region.     

Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin

The influence of various factors on the interaction of phosphorylated and dephosphorylated myosin with actin was examined. It was found that the difference between the values of specific activity of the two myosin forms of actin-stimulated Mg2+-ATPase is affected by changes in KCl, MgATP and actin concentration. The effect of increased pH on the differences in the rate of ATP hydrolysis by actomyosin containing phosphorylated myosin as compared with that of the dephosphorylated one, observed in the presence of EGTA, is abolished by addition of Ca2+. Tropomyosin strongly inhibits the actin-stimulated Mg2+-ATPase of phosphorylated myosin (by about 60%). The tropomyosin-troponin complex and native tropomyosin lowered the rate of ATP hydrolysis by actomyosin containing both phosphorylated and dephosphorylated myosin by about of 60% of the value obtained in the absence of those proteins. These results indicate that the change of negative charge on the myosin head due to phosphorylation and dephosphorylation of myosin light chains modulates the actin-myosin interaction at different steps of the ATP hydrolysis cycle. Phosphorylation of myosin seems to be a factor decreasing the rate of ATP hydrolysis by actomyosin under physiological conditions.     

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.     

Product inhibition studies of yeast phosphoglycerate kinase evaluating properties of multiple substrate binding sites.

Product inhibition studies on yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2- and two for 3-P-glycerate. 2. There is one catalytic centre per enzyme molecule. 3. The catalytic reaction primarily occurs at the 'first' or 'high affinity' MgATP2- and 3-P-glycerate binding sites. The 'second' set of sub-sites for these substrates are located in a region for regulation of the catalytic reaction. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. MgATP2- and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2- the apparent Ki value for 1,3-P2-glycerate increases from 3 microM to 20 microM.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.

ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.     

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis.

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.     

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.     

Thermodynamics of nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong ADP binding

We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.     

Construction and characterization of a heterodimeric iron protein: defining roles for adenosine triphosphate in nitrogenase catalysis

One molecule of MgATP binds to each subunit of the homodimeric Fe protein component of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P(i) in reactions coupled to the transfer of one electron into the MoFe protein component. As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn substituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separation of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction of a seven His tag on one subunit and a differential immobilized-metal-affinity chromatography technique. Three forms of the Fe protein (wild-type homodimeric Fe protein [Asp(39)/Asp(39)], altered homodimeric Fe protein [Asn(39)/Asn(39)], and heterodimeric Fe protein [Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleotide binding. Among those features examined were the MgATP- and MgADP-induced protein conformational changes that are manifested by the susceptibility of the [4Fe-4S] cluster to chelation and by alterations in the electron paramagnetic resonance, circular dichroism, and midpoint potential of the [4Fe-4S] cluster. The results indicate that changes in the [4Fe-4S] cluster caused by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The [Asp(39)/Asn(39)] Fe protein did not support substrate reduction activity but did hydrolyze MgATP and showed MgATP-dependent primary electron transfer to the MoFe protein. These results support a model where each MgATP site contributes to the rate acceleration of primary electron transfer, but both MgATP sites must be functioning properly for substrate reduction. Like the altered homodimeric [Asn(39)/Asn(39)] Fe protein, the heterodimeric [Asp(39)/Asn(39)] Fe protein was found to form a high affinity complex with the MoFe protein, revealing that alteration on one subunit is sufficient to create a tight complex.     

Reaction of two heads of gizzard myosin with ATP

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites

31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Nitrogenase of Klebsiella pneumoniae: an MgATP hydrolysing energy transduction system with similarities to actomyosin and p21 ras.

The mechanism of ATP hydrolysis by nitrogenase shows some similarity to that proposed for actomyosin and for GTP hydrolysis by p21 ras. All three systems involve the formation of an active complex from two component proteins, nucleotide-induced changes in protein conformation, energy transduction that in the case of nitrogenase involves a decrease in redox potential of metal centres, and a slow dissociation of the protein complex. Metal ion activation (Mg2+ or Ca2+) and in-line displacement of ADP by H2O without enzyme phosphorylation are also common features. At 5 degrees C, stopped-flow calorimetry shows that the kinetic and thermodynamic parameters for endothermic, reversible on-enzyme cleavage of MgATP by nitrogenase and myosin subfragment 1 are remarkably similar. [18O4]Pi-water exchange studies also show that ATP cleavage on nitrogenase and myosin are reversible.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

The effect of magnesium ions on the two-stage kinetics of superprecipitation and ATPase activity of natural actomyosin

The effect of magnesium ions on the two-stage kinetics of superprecipitation (SPP) and ATP activity of natural skeletal muscle actomyosin was studied. It was found that the changes in the ratios of two independent steps of SPP and ATPase activity are mainly induced by the Mg-ATP2- complex, but not by free Mg2+. These changes in the kinetics of SPP and ATPase are regarded as being due to the shift in the dynamic equilibrium between the two types of the actomyosin complexes in solution, each of which is characterized by different reaction mechanisms. The role of the Mg-ATP2(-)-induced alteration of at least two structural-and-functional states of actomyosin in muscle contractibility is discussed.