Role of AMP deaminase reaction in the control of fructose 1,6-bisphosphatase activity in yeast

The physiological role of the inhibition of AMP deaminase (EC 3.5.4.6) by Pi was analyzed using permeabilized yeast cells. (a) Fructose 1,6-bisphosphatase (EC 3.1.3.11) was inhibited only a little by AMP, which was readily degraded by AMP deaminase under the in situ conditions. (b) The addition of Pi, which showed no direct effect on fructose 1,6-bisphosphatase, effectively enhanced the inhibition of the enzyme by AMP increased through the inhibition of AMP deaminase. (c) Pi activated phosphofructokinase (EC 2.7.1.11) and inhibited AMP deaminase activity. AMP deaminase reaction can act as a control system of fructose 1,6-bisphosphatase activity and gluconeogenesis/glycolysis reaction through the change in the AMP level. Pi may contribute to the stimulation of glycolysis through the inhibition of fructose 1,6-bisphosphatase by the increase in AMP in addition to the direct activation of phosphofructokinase.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.     

Involvement of threonine deaminase in repression of the isoleucine-valine and leucine pathways in Saccharomyces cerevisiae

l-Threonine deaminase (l-threonine dehydratase [deaminating], EC 4.2.2.16) has been shown to be involved in the regulation of three of the enzymes of isoleucine-valine biosynthesis in yeast. Mutations affecting the affinity of the enzyme for isoleucine also affected the repression of acetohydroxyacid synthase, dihydroxyacid dehydrase, and reductoisomerase. The data indicate that isoleucine must be bound for effective repression of these enzymes to take place. In a strain with a nonsense mutation midway in liv 1, the gene for threonine deaminase, starvation for isoleucine or valine did not lead to derepression of the three enzymes; starvation for leucine did. The effect of the nonsense mutation is recessive; it is tentatively concluded, therefore, that intact threonine deaminase is required for derepression by two of the effectors for multivalent repression, but not by the third. A model is presented which proposes that a regulatory species of leu tRNA(leu) is the key intermediate for repression and that threonine deaminase is a positive element, regulating the available pool of charged leu tRNA by binding it.     

Complementation of a threonine dehydratase-deficient Nicotiana plumbaginifolia mutant after Agrobacterium tumefaciens-mediated transfer of the Saccharomyces cerevisiae ILV1 gene.

The Saccharomyces cerevisiae ILV1 gene, encoding threonine dehydratase (EC 4.2.1.16) was fused to the transferred DNA nopaline synthase promoter and the 3' noncoding region of the octopine synthase gene. It was introduced, by Agrobacterium tumefaciens-mediated gene transfer, into an isoleucine-requiring Nicotiana plumbaginifolia auxotroph deficient in threonine dehydratase. Functional complementation by the ILV1 gene product was demonstrated by the selection of several transformed lines on a medium without isoleucine and by the identification in these lines of the yeast threonine dehydratase activity. This is the first example illustrating the complementation of a plant auxotroph by transfection with a cloned gene.     

Purification and properties of threonine deaminase from the X-1 isolate of the genus Thermus

Threonine deaminase (l-threonine dehydratase EC 4.2.1.16) has been partially purified from a new extreme thermophilic bacterium, Thermus X-1, which is similar to T. aquaticus YT-1. The threonine deaminase of strain X-1 has a maximal rate of reaction at 85 to 90 C and is more thermostable than the threonine deaminase from mesophilic bacteria. The enzyme has an apparent molecular weight of 100,000 to 115,000, a K(m) for l-threonine of 14 mM, a pH optimum of 8.0, and like other threonine deaminases also catalyzes the deamination of serine. However the Thermus X-1 threonine deaminase does not show a strong feedback inhibition by isoleucine. It is suggested that the regulation of the biosynthesis of isoleucine in this extreme theromophile may resemble that reported in Rodospirillum rubrum.     

AMP deaminase as a control system of glycolysis in yeast. Mechanism of the inhibition of glycolysis by fatty acid and citrate

The role of fatty acid and citrate on the interaction of the AMP deaminase (EC 3.5.4.6) reaction with glycolysis was investigated using permeabilized yeast cells. (a) Linolenate and citrate inhibited glycolytic flux and the recovery of the adenylate energy charge; however, linolenate remarkably retarded the depletion of the total adenylate pool, which was not at all affected by the addition of citrate. (b) Linolenate inhibited AMP deaminase activity in situ, resulting in the subsequent decrease in ammonium production, which reduced the activity of 6-phosphofructokinase (EC 2.7.1.11), whereas linolenate itself had no ability to inhibit the phosphofructokinase activity in the presence of excess ammonium concentration. (c) Citrate inhibited the activity of phosphofructokinase in situ in the presence and absence of ammonium ion, followed by an inhibition of glycolysis; however, AMP deaminase activity was not inhibited by citrate. The inhibition of glycolysis by fatty acids can be accounted for by the lowered activity of phosphofructokinase as a result of the decreased level of ammonium ion through the inhibition of the AMP deaminase reaction by these ligands, whereas the effect of citrate on glycolysis is a direct inhibition of phosphofructokinase without affecting the activity of AMP deaminase. Fatty acid and citrate, a principal metabolic product of fatty acid oxidation, can be responsible for the control of glycolysis in two different manners.     

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.     

AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge

The role of ammonium ion and AMP deaminase (EC 3.5.4.6) reaction in the activation of phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) by the decrease in the adenylate energy charge was investigated using permeabilized yeast cells. Response of AMP deaminase, phosphofructokinase, and pyruvate kinase to variation in the energy charge is typical of the ATP-regenerating enzymes: an activation with the decrease in the energy charge under the in situ conditions. The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the phosphofructokinase activity with the increase in the optimal energy charge value giving maximal activity of the enzyme. The optimal energy charge value of phosphofructokinase was 0.2-0.25 in the absence of ammonium ion and was shifted to the value above 0.5 by the addition of ammonium ion, whereas Pi, an activator of the enzyme showed little effect on the increase in the optimal energy charge value. The optimal energy charge value of AMP deaminase and pyruvate kinase was not affected by the addition of their effectors. Modulation of the response to the energy charge of phosphofructokinase and pyruvate kinase was analyzed in terms of the "activation coefficient," which was defined as the ratio of the activity at the energy charge of 0.6 to that at the value of 0.9. Activation of phosphofructokinase by the physiological decrease in the energy charge (0.9 to 0.6) can be enhanced by the increase in ammonium ion specifically, although the coefficient of pyruvate kinase remained unaffected by ammonium ion. These results suggest that the AMP deaminase reaction as an ammonium-forming reaction can participate in a key role in the stimulation of phosphofructokinase or glycolytic flux in cells.     

Synthesis of biodegradative threonine dehydratase in Escherichia coli: role of amino acids, electron acceptors, and certain intermediary metabolites

The specific activity of inducible biodegradative threonine dehydratase (EC 4.2.1.16) in Escherichia coli K-12 increased significantly when the standard tryptone-yeast extract medium or a synthetic mixture of 18 L-amino acids was supplemented with 10 mM KNO3 or 50 mM fumarate and with 4 mM cyclic AMP. In absolute terms, almost four times as much enzyme was produced in the amino acid medium as in the tryptone-yeast extract medium. Enzyme induction in the amino acid medium was sensitive to catabolite repression by glucose, gluconate, glycerol, and pyruvate. An analysis of amino acid requirements for enzyme induction showed that a combination of only four amino acids, threonine, serine, valine, and isoleucine, produced high levels of threonine dehydratase provided that both fumarate and cyclic AMP were present. Immunochemical data revealed that the enzyme synthesized in the presence of these four amino acids was indistinguishable from that produced in the tryptone-yeast extract or the medium with 18 amino acids. We interpret these results to mean that not the amino acids themselves but some metabolites derived anaerobically in reactions involving an electron acceptor may function as putative regulatory molecule(s) in the anaerobic induction of this enzyme.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Subcellular Localization of Isoleucine-Valine Biosynthetic Enzymes in Yeast

By using the method of stepwise homogenization of yeast spheroplast lysates employed previously with the leucine biosynthetic enzymes, it is shown that threonine deaminase, acetohydroxy acid synthase, Mg(2+)-dependent isomero-reductase, and dihydroxy acid dehydratase are particulate. Density gradient centrifugation and the behavior of marker enzymes suggest that all of the above enzymes of the isoleucine-valine biosynthetic pathway are associated with the mitochondria.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.     

Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli.

To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.     

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.     

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP

The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.     

A kinetic study of the inhibition of yeast AMP deaminase by polyphosphate

Inorganic pyrophosphate and polyphosphates have acted as potent inhibitors of purified AMP deaminase (EC 3.5.4.6) from yeast: the activity fell to a definite limit with the increase in the concentration of the inhibitor. The effect of polyphosphate was largely on the maximal velocity of the enzyme with some decrease in affinity. The cooperative effect of AMP, analyzed in terms of a Hill coefficient, remained at 2 in the absence and presence of polyphosphate. Binding of polyphosphate to the enzyme showed no cooperativity. The inhibition of AMP deaminase by polyphosphate can be qualitatively and quantitatively accounted for by the partial mixed-type inhibition mechanism. Both the Ki value for the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of polyphosphate, suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by binding of polyphosphate to a specific inhibitory site.     

The effect of high protein diet on the regulatory properties of AMP deaminase from chicken liver

Feeding high protein diet for 5 days caused a 3,5-fold and 2-fold increase of the activity of xanthine dehydrogenase (EC 1.2.1.37) and 5-nucleotidase (EC 3.1.3.31) respectively, in chicken liver. Six hours after feeding the high protein diet there was no change in either enzyme activity although a 3-fold increase in the level of serum uric acid was observed. High protein diet considerably decreased the activity of AMP deaminase at low, but not at high substrate concentration. The activity ratio, measured at 10.0 and 0.16 mM AMP increased from 14:1 (low protein diet) to 23:1 and 24:1 after 6 h and 5 days of high protein diet, respectively. It has been suggested that feeding birds a high protein diet may cause transformation of liver AMP deaminase (EC 3.5.4.6) from a low Km form toward a high Km form.     

Effects of various ligands on interaction of AMP deaminase with myosin.

Purified rat muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) binds tightly to rat myosin. The binding is abolished in the presence of low concentrations of various ligands. Pyrophosphate and GTP at concentrations as low as 0.1 micrometer were effective in abolishing the interaction between two proteins. Other nucleoside triphosphates were less effective than GTP and the concentrations required for 50% inhibition were approximately 0.3 to 0.7 micrometer. ADP and AMP are effective in inhibiting the interaction between two proteins, but they are less effective than the nucleoside triphosphates; 50% inhibition occurred at 34 micrometer with ADP and at 1 mM with AMP. Creatine phosphate and inorganic phosphate showed 50% inhibition at 5 to 6 mM. All of the compounds, which affected AMP deaminase activity, were effective in abolishing the interaction of the enzyme with myosin; however, the interaction-abolishing effects of the compounds are not parallel with their inhibitory effects on the deaminase activity. Although there exist three parental isozymes of AMP deaminase in the rat, all three enzymes interacted with myosin.