Anomalous oxygen-18 exchange during ATP synthesis in oxidative phosphorylation

The synthesis of ATP from highly enriched [18O]Pi by submitochondrial particles driven by succinate oxidation produces distributions of 18O-labeled ATP species that deviate from the distributions predicted by a simple model for the exchange. Control experiments indicate no change in isotopic distribution when [18O]ATP is synthesized from [18O]ADP by adenylate kinase, which is bound to the submitochondrial particles. The observed deviations are in the opposite direction from that produced by heterogeneity due to multiple pathways for ATP synthesis. Two types of complex models can account for the observed deviations. One model has nonequivalence of the Pi oxygens during the exchange reaction, due to incomplete randomization of the Pi oxygens during the reversible cycles of hydrolysis and synthesis of bound ATP. The other model assumes that, during each turnover, a slow transition must occur between a high-exchange and a low-exchange pathway.     

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.     

Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges

We have examined intermediate Pi-water oxygen exchange during [gamma-18O]ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12. Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources. Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules. The epsilon-replete enzyme showed a much higher probability for oxygen exchange. These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release. Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis. Heterogeneity was apparent in the 18O distribution of the product Pi, however. That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit. In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP. This is the only enzyme so far reported with this unusual property. Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.     

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.     

Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.     

Measurement of the reversibility of ATP binding to myosin in calcium-activated skinned fibers from rabbit skeletal muscle. Oxygen exchange between water and ATP released to the solution

We have measured the rate constant for ATP release from myosin heads of Ca2+-activated, demembranated muscle fibers using the technique of phosphate-water oxygen exchange. Single rabbit psoas fibers were held in an activating solution in [18O]water ([MgATP] = 8 mM, ionic strength = 0.2 M, pH = 7.0, 24 degrees C). After about 20% hydrolysis of ATP, product Pi and remaining ATP were isolated, and the distribution of 18O in both molecules was analyzed using a mass spectrometer. The exchange in Pi was similar to that previously reported (Hibberd, M. G., Webb, M. R., Goldman, Y. E., and Trentham, D. R. (1985) J. Biol. Chem. 260, 3496-3501). The amount of 18O in ATP gave a rate constant of about 4 s-1 for ATP release, if it is assumed that each rate constant in the pathway of ATP hydrolysis has the same value for all myosin ATPase sites. However, the distribution of 18O in both released Pi and ATP is not well explained by a single pathway for ATP hydrolysis. We present a model that indicates how such distributions could arise from a range of values for the rate constants for Pi and ATP release from actomyosin, and this range is determined by differences in the amounts of strain in attached crossbridges. The kinetic information obtained from these isotope exchange experiments is compared to show that they give a compatible set of rate constants for actomyosin in fibers.     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.     

Characterization of phosphate oxygen exchange reactions catalyzed by myosin through measurement of the distribution of 18-O-labeled species

The change in the distribution of the phosphate species containing 0 to 4 18O oxygens per Pi was investigated during medium Pi equilibrium HOH exchange catalyzed by myosin subfragment 1. At 25 degrees C, a Pi molecule once bound loses an average of 3.9 of its original 4 oxygens prior to release which means that at least 100 reversals of the exchange reaction must have occurred. At 0 degrees C, only 3.4 of the 4 oxygens are lost prior to release indicating an average of 17 reversals. Distribution patterns are consistent with equivalent participation in the exchange reactions of all 4 oxygens of bound Pi. The intermediate exchange of Pi oxygens during hydrolysis of 18O-labeled ATP by myosin has also been investigated. The distribution of the product Pi species shows that there is an ATPase component in myosin preparations which hydrolyzes ATP without intermediate exchange. Presence of this component, which is likely a contaminating ATPase, provides a simple explanation of the apparent nonequivalence of phosphate oxygens which has been observed. When correction is made for this contaminant, characteristics of the myosin intermediate Pi equilibrium HOH exchange are similar to those of myosin subfragment 1 medium exchange, and intermediate exchange data are in much closer agreement with other kinetic measurements.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F1 with Mg-ATP was required for the binding of the natural inhibitor, IF1, to F1 to form the inactive F1-IF1 complex. When F1 was incubated in the presence of [14C]ATP and MgCl2, about 2 mol 14C-labeled adenine nucleotides were found to bind per mol of F1; the bound 14C-labeled nucleotides consisted of [14C]ADP arising from [14C]ATP hydrolysis and [14C]ATP. The 14C- labeled nucleotide binding was not prevented by IF1. These data are in agreement with the idea that the formation of the F1-IF1 complex requires an appropriate conformation of F1. (2) The 14C-labeled adenine nucleotides bound to F1 following preincubation of F1 with Mg-[14C] ATP could be exchanged with added [3H]ADP or [3H]ATP. No exchange occurred between added [3H]ADP or [3H]ATP and the 14 C-labeled adenine nucleotides bound to the F1-IF1 complex. These data suggest that the conformation of F1 in the isolated F1-IF1 complex is further modified in such a way that the bound 14C-labeled nucleotides are no longer available for exchange. (3) 32Pi was able to bind to isolated F1 with a stoichiometry of about 1 mol of Pi per mol of F1 (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899). There was no binding of 32Pi to the F1-IF1 complex. Thus, not only the nucleotides sites, but also the Pi site, are masked from interaction with external ligands in the isolated F1-IF1 complex.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence from 18O exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric H+, K+-ATPase by inorganic phosphate

Phosphorylation of the gastric H,K-ATPase by Pi has been studied by measuring the P18Oj16O4-j distribution as a function of time at different H+, K+, and [18O]Pi concentrations. The advantage of isotope exchange measurements is that the P18Oj16O4-j distribution depends on the relative rates of HOH loss to form the phosphoenzyme intermediate and Pi dissociation from the enzyme. Therefore, 18O exchange is a sensitive probe of mechanism. K+ increases the exchange rate (v(ex] but does not affect the partition coefficient (Pc) that determines the P18Oj16O4-j distribution. Conversely, H+ inhibits exchange. A single Pc describes the data at every pH, but the value increases from 0.04 at pH 8 to 0.64 at pH 5.5. Vex depends hyperbolically on [Pi]0. Km for Pi does not depend on pH, and Pc does not depend on [Pi]0. Individual rate constants in the phosphorylation mechanism are estimated. Formation of the E.Pi complex that looses HOH is 1-2 orders of magnitude slower at pH 5.5 than at pH 8 and is not diffusion controlled. The observed change in Pc with pH is compatible with catalysis occurring by a different mechanism when a group with pKa = 7.2 is protonated. Slower than diffusion-controlled formation of the E.Pi complex that splits out HOH is evidence for a relatively slow, unimolecular chemical transformation involving an additional intermediate in the phosphorylation mechanism, such as a protein conformational change.     

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.     

Kinetic evidence for the formation of D-alanyl phosphate in the mechanism of D-alanyl-D-alanine ligase

The steady state kinetic mechanism, molecular isotope exchange and the positional isotope exchange (PIX) reactions of D-alanyl-D-alanine ligase from Salmonella typhimurium have been studied. The kinetic mechanism has been determined to be ordered Ter-Ter from initial velocity and product inhibition experiments. The first substrate to bind is ATP followed by the addition of 2 mol of D-alanine. Pi is released, and then D-alanyl-D-alanine and ADP dissociate from the enzyme surface. In the reverse direction D-alanyl-D-alanine exhibits complete substrate inhibition (Ki = 1.15 +/- 0.05 mM) by binding to the enzyme-ATP complex. In the presence of D-alanine, D-alanyl-D-alanine ligase catalyzed the positional exchange of the beta,gamma-bridge oxygen in [gamma-18O4]ATP to a beta-nonbridge position. Two possible alternate dead-end substrate analogs, D-2-chloropropionic acid and isobutyric acid, did not induce a positional isotope exchange in [gamma-18O4]ATP. The positional isotope exchange rate is diminished relative to the net substrate turnover as the concentration of D-alanine is increased. This is consistent with the ordered Ter-Ter mechanism as determined by the steady state kinetic experiments. The ratio of the positional isotope exchange rate relative to the net chemical turnover of substrate (Vex/Vchem) approaches a value of 1.4 as the concentration of D-alanine becomes very small. This ratio is 100 times larger than the ratio of the maximal reverse and forward chemical reaction velocities (V2/V1). This situation is only possible when the reaction mechanism proceeds in two distinct steps and the first step is much faster than the second step. The enzyme was also found to catalyze the molecular isotope exchange of radiolabeled D-alanine with D-alanyl-D-alanine in the presence of phosphate. These results are consistent with the formation of D-alanyl phosphate as a kinetically competent intermediate.     

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.     

Study of dynein ATPase by oxygen isotope exchange H2(18)O in equilibrium with [16O]-Pi

The oxygen isotope exchange reactions catalyzed by sea urchin Strongylocentrotus intermedius spermatozoa dynein I were studied with a view of comparing molecular mechanisms of ATP hydrolysis by dynein and myosin ATPases. It was demonstrated that the isotope exchange takes place during ATP hydrolysis and during enzyme incubation with ADP and Pi and is absent when the enzyme is incubated with Pi. It was assumed that the molecular mechanisms of ATP hydrolysis by dynein I and myosin are identical.     

Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis

In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.     

Rate of ATP synthesis by dynein

The rates of ATP synthesis and release by the dynein ATPase were determined in order to estimate thermodynamic parameters according to the pathway: (Formula: see text). Dynein was incubated with high concentrations of ADP and Pi to drive the net synthesis of ATP, and the rate of ATP production was monitored fluorometrically by production of NADPH through a coupled assay using hexokinase and glucose-6-phosphate dehydrogenase. The turnover number for the rate of release of ATP from 22S dynein was 0.01 s-1 per site at pH 7.0, 28 degrees C, assuming a molecular weight of 750 000 per site. The same method gave a rate of ATP synthesis by myosin subfragment 1 of 3.4 X 10(-4) s-1 at pH 7.0, 28 degrees C. The rate of ATP synthesis at the active site was estimated from the time dependence of medium phosphate-water oxygen exchange. Dynein was incubated with ADP and [18O] Pi, and the rate of loss of the labeled oxygen to water was monitored by 31P NMR. A partition coefficient of 0.31 was determined, which is equal to k-2/(k-2 + k3). Assuming k3 = 8 s-1 [Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13832], k-2 = 3.5 s-1. From the rates of ATP binding and hydrolysis measured previously (Johnson, 1983), the equilibrium constants for ATP binding and hydrolysis could be calculated: K1 = 5 X 10(7) M-1 and K2 = 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate-limiting step in the actomyosin adenosinetriphosphatase cycle: studies with myosin subfragment 1 cross-linked to actin

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)