Folic acid and pterin deaminases in Dictyostelium discoideum: kinetic properties and regulation by folic acid, pterin, and adenosine 3'',5''-phosphate.

Kinetic data obtained for deamination of pterin by the extracellular fraction from Dictyostelium discoideum yielded apparently linear Lineweaver-Burk plots for pterin. The Michaelis constant for pterin was 30 microM. The data for folic acid deamination yielded convex Lineweaver-Burk plots. Convex Lineweaver-Burk plots could result from the presence of two types of enzymes with different affinities. The data for folic acid deamination were analyzed mathematically for two types of enzymes. This analysis produced Michaelis constants for folic acid of 1.8 and 23 microM competition studies suggested that an enzyme with low affinity nonspecifically catalyzed the deamination of folic acid and pterin, whereas an enzyme with high affinity was a specific folic acid deaminase. A specific folic acid deaminase with high affinity appeared to be present on the surface of D. discoideum cells. The Michaelis constant for this enzyme was 2.6 microM. Cells growing in nutrient broth and cells starved in phosphate buffer released folic acid and pterin deaminases. The quantity of deaminase activities released by the cells appeared to be controlled by chemoattractants. Starving cells that were supplied with folic acid, pterin, or adenosine 3',5'-phosphate increased their extracellular folic acid and pterin deaminase activities to a larger extent than did cell suspensions to which no chemoattractants were added. Administration of folic acid or pterin to starving cells caused increases of the activity of extracellular adenosine 3',5'-phosphate phosphodiesterase and repressed increases of the activity of phosphodiesterase inhibitor.     

Developmental changes of chicken liver AMP deaminase.

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1-7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.     

Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.     

Comparison of kinetic and regulatory properties of high S0.5 form of AMP deaminase from chicken and pigeon liver with AMP deaminase from rat and ox liver

1. The high S0.5 form of AMP deaminase from avian liver was shown to display a two times lower S0.5 value than the single mammalian enzyme form. 2. Avian enzymes showed several fold higher affinity to the activator (ATP) but lower affinity to inhibitors (GTP and Pi) than the mammalian AMP deaminases. 3. GTP was shown to exert a biphasic: activating and inhibitory effect on all the enzymes tested, the chicken and pigeon enzymes being activated within a much broader range of effector concentration. 4. In the presence of 3 mM ATP the activity of avian enzymes was not affected by high GTP and Pi concentrations, in contrast to AMP diaminase from rat liver which was strongly inhibited by GTP under the same experimental conditions. 5. The differences of the regulatory properties described are discussed in terms of adjustment of avian liver AMP deaminase to a faster adenylates' catabolism and thus urate synthesis.     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.     

Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.     

Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase.

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.     

A specific AMP deaminase assay and its application to tissue homogenates

1. A rapid method for the determination of AMP and IMP by HPLC is described. 2. Its application to the assay of AMP deaminase allows the specific determination of enzyme activities in crude extracts, eliminating any interference by other enzyme systems (5'-nucleotidase and adenosine deaminase). 3. The method was routinely used for the determination of the AMP deaminase activity in the muscles of marine animals.     

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.     

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.     

Sub-families of alpha/beta barrel enzymes: a new adenine deaminase family

No gene coding for an adenine deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold.     

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.     

A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography.

A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Growth stimulation of sparse, serum deprived and of confluent, contact inhibited mammalian fibroblasts by a preparation of 3':5'-cyclic AMP phosphodiesterase.

Resting mammalian fibroblasts, either sparse and maintained in a serum-free medium, or confluent and contact inhibited, are stimulated to divide by treatment with a preparation of 3':5'-cyclic AMP phosphodiesterase. This enzyme preparation contained a low level of trypsin-like and alpha-chymotrypsin-like activity, but its effect on cell growth could not be mimicked by pure, crystallized trypsin and alpha-chymotrypsin at concentration equivalent to their contamination in the above preparation. Preincubation of the 3':5'-cyclic AMP phosphodiesterase preparation with the protease inhibitor, phenyl methane sulfonyl fulride, did not affect, either, its stimulation of DNA synthesis in fibroblasts, or its enzymatic hydrolysis of cyclic AMP.     

AMP deaminase from the gill of Salmo gairdnerii Richardson: effects of anions, cations and buffers

The AMP deaminase isoenzymes from trout gill were activated by sodium and potassium, sodium being the most efficient. The optimal concentration for activation was 30-50 mM. The enzyme was sensitive to ionic strength, and imidazole was an inhibitor at concentrations higher than 25 mM. A possible regulation of gill AMP deaminase by intracellular imidazole buffers is discussed. AMP deaminase activity was tested in the presence of physiological concentrations of sodium and potassium. When the concentration of one of these cations was varied around its physiological concentration, the enzyme activity was relatively stable, indicating that the intracellular AMP deaminase activity would be insensitive to changes in the concentrations of monovalent cations. The effects of the sodium salts of different inorganic and organic anions were tested. Except chloride and gluconate, all were inhibitors of gill AMP deaminase.     

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

Isolation and regulation of piglet cardiac AMP deaminase

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a K_m of 2 mM for 5-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the K_m of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD⁺, whereas, the K_m was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in V_max with no change in K_m. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.