Negative homotropic cooperativity in rat muscle AMP deaminase. A kinetic study on the inhibition of the enzyme by ATP

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Regulatory properties of 14-day embryo and adult hen skeletal muscle AMP deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of 14 day-old chicken embryo and adult hen skeletal muscle AMP deaminase. In the presence of 5 mM potassium the enzyme from embryo muscle exhibited a sigmoid-shaped plot of the reaction rate versus substrate concentration. The increase of KCl concentration up to 100 mM diminished distinctly sigmoidicity of the plot. Micromolar concentrations of ADP or ATP activated, whereas GTP at the same concentrations inhibited the embryo and hen skeletal muscle AMP deaminase while 5 mM KCl was present in the incubation medium. 100 mM potassium concentration diminished the effect of ADP and ATP but not of GTP. Palmitoyl-CoA inhibited strongly the embryo skeletal muscle adenylate deaminase but had no effect on the activity of the hen enzyme. Alanine inhibited only the adult hen enzyme. The embryo and hen AMP deaminase differed also in the specificity to adenylate analogues and exhibited a different dAMP/AMP ratio. The data presented indicate that kinetic and regulatory properties of the two developmental forms of AMP deaminase are different.     

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.     

AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.     

Nuclear-membrane-associated protein kinase and substrates. The effect of growth state on activity and specificity.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.     

Purification and general properties of AMP deaminase from sheep brain

AMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350,000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85,000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100 mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase from human liver.     

Interaction of rat muscle AMP deaminase with myosin. II. Modification of the kinetic and regulatory properties of rat muscle AMP deaminase by myosin.

The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP.     

Covalent modification of the inhibitor binding site(s) of Escherichia coli ADP-glucose synthetase: specific incorporation of the photoaffinity analogue 8-azidoadenosine 5'-monophosphate

The photoaffinity agent 8-azidoadenosine 5'-monophosphate (8-N3AMP) is an inhibitor site specific probe of the Escherichia coli ADP-glucose synthetase (ADPG synthetase). In the absence of light, 8-N3AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), the analogue specifically and covalently modifies the enzyme, and photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADPG provides nearly 100% protection from 8-N3AMP photoinactivation, while the substrate ATP provides approximately 50% protection and the inhibitor AMP, approximately 30% protection. These three adenylate allosteric effectors of E. coli ADPG synthetase also protect it from photoincorporation of 8-N3AMP. A structural overlap of the inhibitor and substrate binding sites is proposed which explains the protection data in light of the known binding and kinetic properties of this tetrameric enzyme.     

The role of GTP in the regulation of two forms of AMP deaminase from chicken kidney

1. Two molecular forms of AMP deaminase have been revealed by phosphocellulose column chromatography of the chicken kidney extract. 2. The chromatographic, kinetic and regulatory properties of these two forms were similar to these of two enzyme forms previously found in the chicken liver, lizard liver and in rat small intestine. 3. GTP exerted different effect from MgGTP on the activity and kinetic parameters of both AMP deaminase I and II from chicken kidney.     

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.     

The nature of inactivation of rat muscle 5''-adenylate aminohydrolase by fluorodinitrobenzene.

1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5'-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.     

Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl.

Adenylate deaminase from rat skeletal muscle has been studied with the objective of understanding how the activity of the enzyme is regulated in vivo. ATP and GTP inhibit the enzyme at low concentrations in the presence of 150 mM KCl. The ATP inhibition is reversed as the ATP concentration is raised to physiological levels. The GTP inhibition is reversed as the GTP concentration is raised to unphysiologically high levels. In the presence of physiological concentrations of ATP, the GTP inhibition is also greatly diminished, but inhibition by orthophosphate remains strong. The apparent affinities of the enzyme for GTP, ATP, and orthophosphate are reduced as the pH is decreased from 7.0 to 6.2. ADP also reduces the apparent affinities of the enzyme for the inhibitors. The regulatory effects of GTP, ATP, and ADP are produced primarily by their unchelated forms. Comparison of the kinetic behavior of the enzyme in vitro with metabolite concentrations in vivo indicates that the major variables that regulate the activity of adenylate deaminase of muscle in vivo are the concentrations of AMP, ADP, orthophosphate, and H+.     

Kinetics of the inhibition by naphtholsulfonate compounds of AMP deaminase from chicken erythrocytes

The effect of a variety of naphthalene sulfonate compounds on the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction was analyzed kinetically. Of the naphthalene sulfonate derivatives tested, the compounds with hydroxyl, sulfonate and nitrogen groups such as amino, anilino or azo groups showed an inhibitory effect. The cooperative effect of AMP, analyzed in terms of Hill coefficient, was increased from about 2 to 4 and the maximal velocity was unchanged with the addition of these compounds, suggesting the ligands as an allosteric inhibitor of the enzyme. The inhibition of AMP deaminase by naphtholsulfonate compounds can be qualitatively and quantitatively accounted for by the Monod-Wyman-Changeux model. Theoretical curves yield a satisfactory fit of all experimental saturation and inhibition curves, assuming four binding sites for AMP and the inhibitor, and various KT(I) values. The structure-activity analysis of the interaction of the naphtholsulfonate compounds with AMP deaminase has demonstrated that the affinity of the enzyme for naphtholsulfonates as the inhibitors is correlated with electronic properties of the nitrogen atoms attached to naphthalene moiety: the delocalization of lone electron pair on nitrogen through naphtholsulfonate group makes the compound less basic, resulting in more tight binding of the ligand to the enzyme. Introduction of hydrophobic group to naphtholsulfonate moiety increases the binding affinity for the enzyme, and of the inhibition. These results suggest the location of hydrophobic regions as the allosteric inhibitory sites of the enzyme for the binding of naphtholsulfonate compounds.     

Adenosine triphosphate-activated adenylate deaminase from marine invertebrate animals. Properties of the enzyme from lugworm (Arenicola cristata) body-wall muscle.

Adenylate deaminase (AMP aminohydrolase, EC 3.5.4.6) from lugworm (Arenicola cristata) body-wall muscle was partially purified by extraction in KCl solutions and chromatography on phosphocellulose. Enzyme activity was eluted from the column at two salt concentrations. Both forms show co-operative binding of AMP (Hill coefficient, h, 2.85) with s0.5 values of 20 mM and 15.6 mM. ATP and ADP act as positive effectors lowering h to 1.07 and s0.5 to 2mM. The apparent Ka (activation) for ATP was 1.5mM. GTP is an inhibitor with an apparent Ki of 0.12 mM. In vivo the ATP-activated adenylate deaminase is in the active form and may be regulated by changes in GTP concentrations. Adenylate deaminase may act as a primary ammonia-forming enzyme in ammonotelic marine invertebrates with the purine nucleotide cycle.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.     

Regulatory properties of rat heart AMP deaminase.

The kinetic and regulatory properties of purified rat heart AMP deaminase were investigated. In the presence of 100 mM KCl, the enzyme exhibited a slightly sigmoid-shaped plot of reaction rate, vs. substrate concentration, which shifted to a more hyperbolic form when ATP, ADP or GTP were added. ATP was the most potent activator of the enzyme, whereas GTP at low (less than 0.25 mM) concentrations increased the enzyme activity. The activation effect was negligible at higher concentrations of GTP. The calculated value of K0.5 of approx. 3 mM for unactivated enzyme decrased to approx. 0.6 mM and 1.1 mM when 0.5 mM ATP or 1.5 mM ADP were present in the incubation mixture, respectively. The theoretical model (Monod, J., Wyman, J. and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118) gave a partial explanation of these results.     

Regulation of AMP deaminase from chicken erythrocytes. A kinetic study of the allosteric interactions.

The allosteric properties of AMP deaminase [EC 3.5.4.6] from chicken erythrocytes have been qualitatively and quantitatively accounted for by the concerted transition theory of Monod et al., on the assumption that this enzyme has different numbers of binding sites for each ligand. Theoretical curves yield a satisfactory fit for all experimental saturation functions with respect to activation by alkali metals and inhibition by Pi, assuming that the numbers of binding sites for AMP, alkali metals, and Pi are 4, 2, and 4, respectively. The enzyme was inhibited by concentrations of ATP and GTP below 0.1 and 0.25 mM, respectively, whereas activation of the enzyme was observed at ATP and GTP concentrations above 0.4 and 1.5 mM, respectively. These unusual kinetics with respect to ATP and GTP could be also accounted for by assuming 2 inhibitory and 4 activating sites for each ligand.     

Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle.

AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (Kd) of this binding was 0.06 micrometer. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase. AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.