Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs

The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.     

Kinetic mechanism of glutaminyl-tRNA synthetase from human placenta

The order of interaction of substrates and products with human placental glutaminyl-tRNA synthetase was investigated in the aminoacylation reaction by using the steady-state kinetic methods. The initial velocity patterns obtained from both the glutamine-ATP and glutamine-tRNA substrate pairs were intersecting, whereas ATP and tRNA showed double competitive substrate inhibition. Dead-end inhibition studies with an ATP analog, tripolyphosphate, showed uncompetitive inhibition when tRNA was the variable substrate. The product inhibition studies revealed that PPi was an uncompetitive inhibitor with respect to tRNA. The noncompetitive inhibition by AMP versus tRNA was converted to uncompetitive by increasing the concentration of glutamine from 0.05 to 0.5 mM. These and other kinetic patterns obtained from the present study, together with our earlier finding that this human enzyme catalyzed the ATP-PPi exchange reaction in the absence of tRNA, enable us to propose a unique two-step, partially ordered sequential mechanism, with tRNA as the leading substrate, followed by random addition of ATP and glutamine. The products may be released in the following order: AMP, PPi and then glutaminyl-tRNA. The proposed mechanism involves both a quarternary complex including all three substrates and the intermediary formation of an enzyme-bound aminoacyl adenylate, common to the usual sequential and ping-pong mechanisms, respectively, for other aminoacyl-tRNA synthetases.     

Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.     

Biosynthetic direction substrate kinetics and product inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for the ATP phosphoribosyltransferase reaction in the biosynthetic direction were determined and are consistent with a sequential kinetic mechanism. To hold the fractions of magnesium-complexed substrates and products constant so as to avoid possible distortion of reciprocal velocity plots Mg²⁺ binding constants to the substrates ATP and phosphoribosylpyrophosphate and the product pyrophosphate were measured under assay conditions. Several conformational states of the phosphoribosyltransferase distinguishable by other criteria gave similar substrate kinetic behavior. Product inhibition studies were conducted to elucidate the binding order. Phosphoribosyl-ATP was competitive with respect to ATP and was non-competitive with respect to phosphoribosylpyrophosphate. Pyrophosphate was non-competitive with respect to both substrates. The data are consistent with the ordered Bi-Bi kinetic mechanism with ATP binding first to free enzyme and phosphoribosyl-ATP dissociating last from enzyme-product complexes.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii.

Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.     

Positional isotope exchange analysis of the pantothenate synthetase reaction

Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PP(i) must be free to dissociate from the active site upon formation of the pantoyl-adenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.     

Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica

The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.     

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.     

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.     

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.     

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K_m values of about 160 and 240 micromolar and with V_max values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K_m of about 700 micromolar and a V_max of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K_m values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.     

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus

Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.     

Reaction path of phosphofructo-1-kinase is altered by mutagenesis and alternative substrates

Escherichia coli phosphofructokinase (PFK) has been proposed to have a random, nonrapid equilibrium mechanism that produces nonallosteric ATP inhibition as a result of substrate antagonism. The consequences of such a mechanism have been investigated by employing alternative substrates and mutants of the enzyme that produce a variety of nonallosteric kinetic patterns demonstrating substrate inhibition and sigmoid velocity curves. Mutations of a methionine residue in the sugar phosphate binding site produced apparent cooperativity in the interaction of fructose 6-phosphate. Cooperativity could also be seen with native enzyme using a poorly binding substrate, fructose 1-phosphate. With an alternative nucleotide, 1-carboxymethyl-ATP, coupled with a mutation that introduced a negative charge in the nucleotide binding site, one could observe substrate inhibition by fructose 6-phosphate and apparent cooperativity in the interaction with nucleotide. Furthermore, the use of a phosphoryl donor, gamma-thiol-ATP, which greatly reduced the catalytic rate, apparently facilitated the equilibration of all binding reactions and eliminated ATP inhibition. These unusual kinetic patterns could be interpreted within the random, steady-state model as reflecting changes in the rates of particular binding and catalytic events.     

Evidence that ecto-nucleoside-triphosphate pyrophosphatase serves in the generation of extracellular inorganic pyrophosphate in human bone and articular cartilage

Extracellular inorganic pyrophosphate (PPi) is important in the regulation of mineralisation of bone, and in the pathogenesis of chondrocalcinosis, an arthritic disease in which calcium pyrophosphate dihydrate crystals form in articular cartilage. Nucleoside-triphosphate pyrophosphatase, which catalyses the formation of PPi, was previously observed at the surface of human articular chondrocytes in culture. A similar enzyme has been identified in osteoblast-like human bone cells in culture, and is active towards purine and pyrimidine nucleoside triphosphates. The enzyme has high affinity for ATP and is located on the cell surface, and thus could serve in the generation of extracellular PPi. Moreover, no other mechanism for the catabolism of small amounts of exogenous ATP is present in human bone cells. Further evidence for ecto-nucleoside-triphosphate pyrophosphatase serving in the generation of extracellular PPi in articular cartilage and bone was obtained by studying the ability of alternative substrates (which do not yield PPi) to inhibit generation of PPi from ATP. In both articular chondrocytes and bone cells, the enzyme exhibited an apparent preference for ATP over dinucleotide and phosphodiester substrates. Some potential inhibitors of the enzyme activity were also studied in both cell types. ADP moderately inhibited the activity but two bisphosphonate drugs were only slightly inhibitory.     

Inhibition of an ecto-ATP-diphosphohydrolase by azide.

Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E-ATPases have been divided into two subfamilies, ecto-ATPases and ecto-ATP-diphosphohydrolases (ecto-ATPDases). In the presence of either Mg2+ or Ca2+, ecto-ATPDases, including proteins closely related to CD39, hydrolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto-ATPases appear to lack these two properties. This report presents the first systematic kinetic study of a purified ecto-ATPDase, the chicken oviduct ecto-ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem. 271, 16323-16331), with respect to ATP and ADP, and azide inhibition. Km values for ATP obtained at pH 6.4 and 7.4 are 10-30 times lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate, divalent ion, and pH. Nearly complete inhibition by 5 mm azide is obtained when MgADP is the substrate and when assays are conducted at pH 6-6.4. Azide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both Vmax and Km for ADP, it does not alter the Km for ATP. These results suggest that the apparent affinity of azide for the E.ADP complex is significantly greater than that for the free enzyme or E.ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.     

Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase-1.

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(β,γ-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.