Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange

The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E X ATP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

Differential inhibition of Pi-ATP exchange in relation to ATP synthesis and hydrolysis by modification of chloroplast thylakoid membranes with glutaraldehyde

Limited modification of thylakoid membranes with glutaraldehyde inhibits the P_i-ATP exchange reaction much more than ATP synthesis or hydrolysis. More extensive modification of the membranes results in the inhibition of all activities of the ATP synthetase, but does not affect electron transport. Limited modification also does not have much effect on the tight binding of [³H]ADP or the ΔpH supported by ATP hydrolysis. The modification affects the catalytic process itself and not the activation of the latent enzyme. Cross-linking between thylakoid polypeptides is observed only after extensive treatment with glutaraldehyde, while limited modification does not result in cross-linking between polypeptides. The differential inhibition of the P_i-ATP exchange relative to ATP hydrolysis can be explained by the decrease in only one of the kinetic rate constants involved in these reactions. However, the relative insensitivity of photophosphorylation to the modification suggests that different enzyme conformations may participate in phosphorylation (light) and ATP hydrolysis or P_i-ATP exchange (dark).     

Microtubules accelerate ADP release by dynein

The effects of microtubules on the phosphate-water oxygen exchange reactions catalyzed by dynein were examined in order to determine the mechanism by which microtubules activate the ATPase. Microtubules inhibited the rate of medium exchange observed during net ATP hydrolysis. Inhibition of the exchange reaction was proportional to the extent of microtubule activation of ATP turnover with no effect on the partition coefficient. These data argue that microtubules do not increase the rate of release of phosphate from dynein; rather, they increase the rate of ADP release. Microtubules markedly inhibited medium phosphate-water exchange reactions observed in the presence of ADP and Pi. With increasing concentrations of ADP, the rate of exchange increased in parallel to the dissociation of dynein from the microtubules, suggesting that only free dynein and not the microtubule-dynein complex catalyzes the exchange reaction. The rates of dynein binding to microtubules in the absence and presence of saturating ADP were 1.6 X 10(6) and 9.8 X 10(5) M-1 s-1, respectively. ADP inhibited the rate of the ATP-induced dissociation of the microtubule-dynein complex with an apparent Kd = 0.37 mM for the binding of ADP to the microtubule-dynein complex. However, the rate of dissociation of ADP from the M.D.ADP complex was quite fast (approximately 1000 s-1). These data support the postulate of a high-energy dynein-ADP intermediate and indicate that microtubules activate the dynein ATPase by enhancing the rate of ADP release.     

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.     

Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation.

Initial velocity and isotope exchange studies confirmed that the over-all reaction, like that catalyzed by pyruvate carboxylase purified from rat liver and chicken liver, was a nonclassical Ping Pong Bi Bi Uni Uni sequence with ATP and HCO3-binding randomly in the Bi Bi partial reaction. Three possible mechanisms for the coupling of ATP hydrolysis and CO2 fixation are considered: (i) Mechanism i, a concerted mechanism without the formation of a kinetically significant or detectable intermediate; (ii) Mechanism ii, activation of the enzyme by ATP to form an activated phosphoenzyme complex which can react with HCO3- by formation of a phosphorylated intermediate. On the basis of other evidence, an activated intermediate containing the ADP moiety was considered improbable. Evidence is presented which indicates that an isotopic exchange between ATP and ADP in the absence of added orthophosphate is not a property of the sheep kidney enzyme. This observation removed the need to postulate either that this exchange is an abortive reaction, or that there is a compulsory formation of a phosphoenzyme intermediate. Two analogues of ADP, alpha,beta-methylene adenosine diphosphate, and adenosine 5'-phosphosulfate, have been used to provide further evidence against Mechanism ii. Both compounds were competitive inhibitors with respect to MgATP2- (Ki values respectively, 0.58 mM and 3.0 mM, compared with 0.17 mM for ADP), but neither could be phosphorylated by the enzyme. Neither analogue could replace ADP in the HCO3-: oxalacetate isotopic exchange reaction, indicating that phosphorylation of ADP is necessary for this exchange to occur, and that Mechanism ii is not applicable. Since Mechanism iii involves formation of a carbonly phosphate intermediate, analogues of this compound, namely, carbamyl phosphate and phosphonacetic acid were used to examine this pathway. The fact that the enzyme catalyzed the synthesis of ATP from ADP and carbamyl phosphate, and that phosphonacetic acid was a noncompetitive inhibitor with respect to MgATP2- (Ki = 0.5 mM) provides strong evidence that a carbonyl phosphate derivative is involved in the reaction mechanism. However, we have not found from initial velocity studies evidence for the formation of this intermediate, and it may therefore have only a transient existence in an essentially concerted reaction.     

Evidence for an acetyl-enzyme intermediate in the action of acetyl-CoA synthetase

Acetyl-CoA synthetase (EC 6.2.1.1) from yeast is a ligase which catalyzes the synthesis of ATP from ADP and acetyl-CoA or acetyl-dephosphoCoA. The enzyme also catalyzes the rapid and reversible transfer of an acetyl group between CoA and dephospho CoA in the absence of the other components of the total ligase reaction. Such transfer is chemically equivalent to a CoA-acetyl-CoA exchange, and points therefore to an acetyl-enzyme intermediate in the transfer (“exchange”) reaction. Since the “exchange” is an intrinsic activity of the enzyme, it seems probable that the acetyl-enzyme mediates the total ligase reaction as well.     

Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.     

Studies on the mechanism of formyltetrahydrofolate synthetase. The Peptococcus aerogenes enzyme

Two conflicting mechanisms have been proposed for formyltetrahydrofolate synthetase (EC 6.3.4.3). Detailed studies with a clostridial enzyme support a sequential mechanism, while a stepwise mechanism with formation of a dissociable intermediate has been proposed for the Peptococcus aerogenes synthetase. However, the data supporting the P. aerogenes mechanism were obtained using synthetase of questionable purity and the results supporting the mechanism could be attributed to contaminating activities. Consequently, uncertainty still exists with regard to the enzyme mechanism. To resolve this uncertainty, the P. aerogenes formyltetrahydrofolate synthetase has been purified to homogeneity and used in experiments to reinvestigate the reaction mechanism. The results of P1:ATP, ADP:ATP, and formate:10-formyltetrahydrofolate exchange experiments as well as a steady state kinetic analysis revealed no difference in the mechanisms of the P. aerogenes or clostridial synthetases. The results are inconsistent with a stepwise mechanism involving a dissociable intermediate and consistent only with a sequential mechanism.     

The isotope-exchange reactions of ox heart phosphofructokinase

1. Ox heart phosphofructokinase catalyses isotope-exchange reactions at pH6.7 between ADP and ATP, and between fructose 6-phosphate and fructose 1,6-diphosphate, the latter reaction being absolutely dependent on the presence of the magnesium complex of ADP. 2. The reaction kinetics are hyperbolic with respect to substrate concentration for both exchange reactions (within the experimental error). 3. The influence of pH, AMP and citrate suggests that the fructose 6-phosphate-fructose 1,6-diphosphate exchange is subject to effector control, and is abolished by dissociation of the enzyme. 4. These results are discussed in relation to the reaction mechanism of the enzyme.     

Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations.

The capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

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.     

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.     

ATP synthetase: a mixture of factor A and avidin sensitive ATP-Pi exchange activity

The ATP-P_i exchange activity of highly purified preparations of ‘ATP synthetase’ was inhibited by F₁-antiserum, Pullman inhibitor, azide and also by avidin (See You and Hatefi, 1973). The inhibition produced by the first three was relieved in the presence of ADP, and the avidin sensitivity was lost on pretreatment on the avidin with biotin. It is concluded that the ATP-P_i exchange resulted from the combined action of Factor A and a contaminating avidin-sensitive enzyme. The ADP necessary for the exchange reaction catalyzed by the latter was generated by the ATPase activity of Factor A.     

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.     

Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction.

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.     

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.     

Kinetic properties of arginine phosphokinase from honeybees, Apis mellifera L. (Hymenoptera, Apidae)

The kinetic properties of honeybee arginine phosphokinase (APK), which catalyzes the reaction: Arginine phosphate + ADP + H⁺ ? arginine + ATP, have been studied.In the direction of ATP synthesis, the pH optimum was around pH 7.2 and the activation energy over the range 18–44 °C was about 10,500 cal/mole. The optimum ratio of Mg²⁺:ADP was about 4:1.In the direction of arginine phosphate (AP) synthesis, the enzyme had a pH optimum around pH 8.3. The energy of activation for the reaction over the range 22–39 °C was about 7500 cal/mole. The optimum ratio of Mg²⁺:ATP was about 1:1.The initial velocities of the reactions in the direction of ATP and AP synthesis were measured at varying concentrations of one substrate while the concentration of the other substrate was held constant at several levels. The double reciprocal plots of the data obtained yielded a series of intersecting lines, indicating that the enzyme has a sequential mechanism. Radioisotope exchange experiment showed that arginine phosphokinase did not catalyze ATP ? ADP exchange in the absence of arginine. Product inhibition studies showed that arginine was competitive with AP and noncompetitive with ADP; whereas ATP was competitive with ADP and noncompetitive with arginine. The results from initial velocity, radioisotope exchange, and product inhibition studies suggested that the enzyme has a rapid equilibrium, random mechanism.