Kinetic and allosteric properties of L-threonine-L-serine dehydratase from human liver]

It was shown that low concentrations of ATP (1..10(-4)M) and 10-fold concentrations of AMP (1.10(-3)M) at three constant L-threonine concentrations activated the L-threonine dehydratase activity of L-threonine-L-serine dehydratase from human liver, but had no effect on the L-serine dehydratase activity of this enzyme. Higher concentrations of both nucleotides inhibited the enzyme. The effects of ATP and AMP were specific. The activating and inhibiting effects of various concentrations of ATP and AMP were revealed as changes in the shapes of the curves for the initial reaction rate of the L-threonine dehydratase reaction versus initial substrate concentration. For this reaction the curves were not hyperbolic and were characterized by intermediary plateaux. ATP and AMP also influenced the maximal rate of the enzymatic reaction. Using the desensitization method it was shown that the activating effects of ATP and AMP are of allosteric nature. Thus, human liver L-threonine-L-serine dehydratase is an allosteric enzyme, for which positive allosteric effectors are low concentrations of ATP and AMP and negative allosteric effectors are high concentrations of these nucleotides. A possible mechanism of allosteric regulation of the enzyme under catalysis of the L-threonine dehydratase reaction and the lack of regulation under catalysis of the L-serine dehydratase reaction as well as specificity of the allosteric sites of this enzyme to the two nucleotides and the physiological significance of this process are discussed.     

Purification and some properties of liver adenylylsulfate kinase

Adenylylsulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase, EC 2.7.1.25) has been purified over 1300-fold from rat liver in 10% yield. The enzyme has a molecular weight of 58,000 and is composed of four subunits of equal molecular weight. ATP is an allosteric activator of adenylylsulfate kinase, with a Hill coefficient of 2.2 and a K0.5 of 2.5 mM. Adenosine phosphosulfate is a potent inhibitor of adenylylsulfate kinase, but the adenosine phosphosulfate concentration for maximal reaction is dependent on the ATP concentration. At the physiological levels of ATP the inhibition by adenosine phosphosulfate is not likely to play a role, while the allosteric regulation of adenylylsulfate kinase by ATP may be operative.     

Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate

The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.     

Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90% of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.     

Stimulus-secretion coupling of glucose-induced insulin release. LI. Divalent cations and ATPase activity in pancreatic islets

Pancreatic islets can be viewed as a fuel-sensor organ. The amount of ATP used by the islet cells for the maintenance of adequate Ca2+ gradients across membranes is not known. An indirect approach to this issue consists in the measurement of Ca-ATPase activity. The kinetics of Ca-ATPase in islet homogenates yielded a Km for ATP close to 0.1 mM and two Km values for Ca2+ close to 0.13 and 4-6 microM, respectively. Within limits, the Ca-ATPase appeared as a distinct entity from Mg-ATPase. Several divalent cations, including Mg2+, inhibited the Ca-ATPase activity. Calmodulin also inhibited, significantly albeit modestly Ca-ATPase. The activity of the enzyme was increased at high pH or in the presence of bicarbonate. The reaction velocity at close-to-physiological concentrations of ATP, Ca2+ and H+ suggests that the consumption of ATP by the Ca-ATPase may account for a major fraction of the overall rate of ATP breakdown in intact islets.     

Stability and partial reactions of soluble and membrane-bound sarcoplasmic reticulum ATPase

The Ca-ATPase of sarcoplasmic reticulum was solubilized at pH 6.5 and 30 degrees C using different nonionic detergents, Triton X-100, C12E8, Lubrol PX, or Tween 20. After full solubilization by any of these detergents, the enzyme was unstable (t1/2 = 2-3 min) in the absence of Ca2+. The soluble enzyme was stable in the presence of calcium, half-maximal protection being attained in the presence of 0.2 mM Ca2+. In the absence of Ca2+, stability was restored by addition of co-solvents dimethyl sulfoxide or glycerol. In the presence of 4 mM Ca2+, the progressive addition of nonionic detergents to a medium containing leaky vesicles promoted an increase, up to 3-fold, in the rate of ATP hydrolysis. This was not observed when ITP was used as substrate. The small amount of ADP accumulated in the medium during ATP hydrolysis was sufficient to inhibit the ATPase activity of the membrane-bound enzyme but had no effect on the soluble enzyme. Increasing concentrations of detergent promoted a progressive inhibition of the ATP----Pi exchange reaction. The ATP hydrolysis/synthesis ratio of soluble enzyme was 10 times higher than that of membranous enzyme. Addition of co-solvent restored this ratio to values similar to those obtained with membrane-bound Ca-ATPase. Soluble enzyme prepared from native sarcoplasmic reticulum vesicles was able to catalyze the net synthesis of ATP when phosphorylated by Pi in the presence of dimethyl sulfoxide and then diluted in a medium containing 10 mM CaCl2 and 2 mM ADP. This was not observed when the soluble enzyme was prepared from purified Ca-ATPase. The results suggest that some of the partial reactions of the catalytic cycle of Ca-ATPase are dependent on the hydrophobic environment found in the native membrane. This environment can be mimicked by co-solvents.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

Phosphorylase kinase conformers. Detection by proteases

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).     

Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).     

Spectral and catalytical properties of the sarcoplasmic reticulum Ca-ATPase labeled with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)-carbodiimide

N-Cyclohexyl-N'-(dimethylamino)-carbodiimide (NCD-4) labels three sites in the sarcoplasmic reticulum Ca-ATPase which can be resolved by their spectral properties and by their effects on the catalytical activity of the enzyme. One site is not protectable by Ca2+ ions or by dicyclohexylcarbodiimide and is not essential for catalytical activity. Two Ca2+-protectable sites, whose modification leads to a biphasic inhibition of Ca-ATPase activity, have fluorescence emission maxima at 407 nm and 425 nm. The Ca-ATPase modified by NCD-4 hydrolyses ATP but does not translocate Ca2+ nor does it undergo the conformational changes associated with Ca2+ binding in the native enzyme. High concentrations of Ca2+ induce slow biphasic fluorescence quenching in the Ca-ATPase labeled selectively at the 407-nm site but the signals are largely abolished by modification of the 425-nm site. Both vanadate ions and ATP reverse this Ca2+-induced fluorescence quenching. It is proposed that NCD-4 labels the two high-affinity Ca2+-binding sites of the Sarcoplasmic reticulum Ca-ATPase and that the conformational changes in the modified enzyme may reflect interactions between the two sites.     

Chicken liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular properties.

A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.     

Two Escherichia coli fructose-6-phosphate kinases. Preparative purification, oligomeric structure and immunological studies.

Two isoenzymes of fructose-6-phosphate kinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) are present in Escherichia coli K12. One isoenzyme is allosterically inhibited by phosphoenolpyruvate and activated by nucleoside diphosphates, and is a tetramer composed of four subunits of molecular weight 35 000. A simple method for the purification of this enzyme is reported. Equilibrium dialysis indicates that there are four ATP sites and four GDP sites per tetramer. The second isoenzyme is present in low quantity in wild type bacteria. This enzyme is devoid of allosteric properties. A complete method of purification is described. Determination of its molecular weight under native and denaturing conditions indicates that this protein is a dimer composed of two subunits of molecular weight 36 000. Antisera have been produced against both isoenzymes. The antiserum against one isoenzyme does not cross-react with the other. Discrepancies between our results and those of other workers are discussed.     

Phenothiazine inhibition of calmodulin stimulates calcium-dependent potassium efflux in human red blood cells

Elevation of red blood cell calcium increases the efflux of potassium. The active extrusion of calcium from the red cell is regulated by calmodulin. Phenothiazines bind to calmodulin in a calcium-dependent manner preventing the calmodulin from activating a wide variety of cellular processes. The present study shows that phenothiazines increase the efflux of potassium from red cells incubated with the calcium ionophore A23187. The dose dependent effect of trifluoperazine on potassium efflux correlates with its inhibition of Ca-ATPase activity. The phenothiazine effects are dependent upon ATP in that increases in potassium efflux are not observed in energy depleted cells. In calcium buffered ghosts no direct effect of calmodulin or an antibody to calmodulin can be shown. These data suggest that phenothiazines stimulate calcium-dependent potassium loss indirectly by a drug-induced blockage of the calmodulin-activated Ca-ATPase.     

Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase

The allosteric enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50 years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.     

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.     

A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding

Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.     

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.     

Human erythrocyte 5'-AMP aminohydrolase. Purification and characterization

Human erythrocyte 5'-AMP aminohydrolase has been obtained using phosphocellulose chromatography and affinity chromatography on a GTP-agarose column to yield a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 285,000, and is comprised of four subunits. Since limited quantities of the homogeneous enzyme were available, the kinetic properties of a nonhomogeneous preparation purified about 20,000-fold over the red blood cell lysate by phosphocellulose chromatography were examined. Like the muscle enzyme, it exhibits a sigmoid AMP saturation curve in the absence of activating monovalent cations; a hyperbolic saturation curve is observed in the presence of 0.15 M KCl. Activation by monovalent cations and ATP, and inhibition by Pi, PPi, GDP, GTP, and 2,3-diphosphoglyceric acid were studied in more detail.