AMP Deaminases Research Articles

Use of isotope effects to determine enzyme mechanisms

AbstractIsotope effects are a powerful tool for determining the mechanisms of enzymatic reactions. Methods for measurement of isotope effects are reviewed and the equations that describe observed isotope effects and permit determination of intrinsic isotope effects on the isotope‐sensitive step are presented. Aspartate transcarbamoylase is used as an example for how the kinetic mechanism can be determined by observing the size of an isotope effect as a function of the concentration of another substrate. Cytidine, adenosine and AMP deaminases are used to illustrate determining the relative rates of steps in the mechanism. Determination of chemical mechanism is illustrated by data for malic enzyme, OMP decarboxylase, aspartate transcarbamoylase, L‐ribulose‐5‐P 4‐epimerase, oxalate decarboxylase, tryptophan 2‐monooxygenase and the K58A mutant of aspartate aminotransferase when rescued by ammonia. Determination of transition state structure is illustrated by data for formate dehydrogenase, prephenate dehydrogenase, chorismate mutase, as well as for enzymes that catalyze phosphoryl and acyl transfer. Copyright © 2007 John Wiley & Sons, Ltd.

Design and synthesis of inhibitors of adenosine and AMP deaminases.

Nucleosides and nucleotides which are able to undergo covalent hydration in the aglycone ring system are potential inhibitors of the enzymes adenosine deaminase (ADA) and AMP deaminase, respectively. Calculations of the enthalpy of covalent hydration and of enzyme binding energy have been used to design new inhibitors of ADA. The ribosyl triazolotriazine 16, which was synthesized as a result of these calculations, exists predominantly as the covalent hydrate 18 in water and is a potent inhibitor of mammalian ADA (IC(50) 50 nM).

ChemInform Abstract: Synthesis of C‐Ribosyl 1,2,4‐Triazolo[3,4‐f][1,2,4]triazines as Inhibitors of Adenosine and AMP Deaminases.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Synthesis of C‐Ribosyl Imidazo[2,1‐f][1,2,4]triazines as Inhibitors of Adenosine and AMP Deaminases.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

N 6-cyclopropyl-PMEDAP: a novel derivative of 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) with distinct metabolic, antiproliferative, and differentiation-inducing properties

N 6-Cyclopropyl-PMEDAP (cPr-PMEDAP) is a novel derivative of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP). Its cytostatic activity was found to be 8- to 20-fold more pronounced than that of PMEDAP and equivalent to that of the guanine derivative 9-(2-phosphonylmethoxyethyl)guanine (PMEG) against a variety of tumor cell lines. Unlike PMEDAP, but like PMEG, cPr-PMEDAP was equally cytostatic to wild-type and 9-(2-phosphonylmethoxyethyl)adenine/PMEDAP-resistant variants of the human erythroleukemia K562 and the murine leukemia L1210 cell lines. Also, cPr-PMEDAP and PMEG proved to be equipotent inducers of K562 and rat choriocarcinoma RCHO cell differentiation, whereas the differentiation-inducing activity of PMEDAP was 5- to 25-fold less pronounced. Furthermore, compared to PMEDAP, cPr-PMEDAP and PMEG were 10- to 25-fold more potent in inhibiting the progression of K562 cells through the S phase of the cell cycle, resulting in a marked accumulation of the four 2′-deoxyribonucleoside 5′-triphosphate pools. The biological effects of cPr-PMEDAP, but not PMEDAP, were reversed by the adenylate deaminase inhibitor 2′-deoxycoformycin (dCF). Formation of the deaminated derivative of cPr-PMEDAP (i.e. PMEG) was demonstrated in crude extracts from K562 and L1210 cells and in metabolism studies with radiolabeled cPr-PMEDAP and PMEG. This is the very first example of an acyclic nucleoside phosphonate analogue that is susceptible to deamination. However, cPr-PMEDAP was not recognized as a substrate by purified adenosine deaminase or by adenylate deaminase. These findings might point to an as yet unidentified cellular enzyme, sensitive to dCF but different from the common adenosine and AMP deaminases. Our data demonstrate the superior antiproliferative and differentiation-inducing effects of cPr-PMEDAP on tumor cells, as compared to the parent compound PMEDAP, based on the unique metabolic properties of this novel compound.

Synthesis of C-ribosyl 1,2,4-triazolo[3,4-f ][1,2,4]triazines as inhibitors of adenosine and AMP deaminases

Modified C-nucleosides and nucleotides with an enhanced tendency to undergo covalent hydration are of interest as potential inhibitors of adenosine deaminase (ADA) and AMP deaminase, respectively. In a search for such compounds we have synthesized 6-dimethylamino-3-(β-D-ribofuranosyl)-1,2,4-triazolo[3,4-f][1,2,4]triazine 4 in four steps (42% overall yield) from the readily available allonic acid 6 and the hydrazine 7. The hydrazide 16 derived from 6 and 7 (78%) is converted directly into the cyclised chloro compound 19 (62%) with phosphorus trichloride oxide, followed by dechlorination (96%) and deprotection (90%). Riboside 4 undergoes partial hydration in water to the covalent hydrate 22, and is a modest inhibitor of mammalian ADA (IC50 180 µM).

Synthesis of C-ribosyl imidazo[2,1-f ][1,2,4]triazines as inhibitors of adenosine and AMP deaminases

The 3-β-D-ribofuranoside 6 of the new imidazo[2,1-f][1,2,4]triazine 27 is isomeric and isoelectronic with the nucleoside deaminoformycin 1 which is a good inhibitor of adenosine deaminase (ADA) while its 5′-monophosphate 2 is a good inhibitor of adenosine 5′-monophosphate deaminase (AMPDA). The 6-methylsulfanyl derivative 7 of 6 is synthesized by condensation of the monocyclic 1,2,4-triazine 9 with bromo aldehyde 10, which is accompanied by cyclization to give the protected C-nucleoside 21; the 8-methylsulfanyl group of 21 is removed by replacement by hydrazine and oxidation. The 1,2,4-triazine 9 cyclizes similarly with chloroacetaldehyde or its dimethyl acetal to give 6,8-bis(methylsulfanyl)imidazo[2,1-f][1,2,4]triazine 17, which is converted into the parent heterocycle 27 by two routes, and into mono- and di-substituted derivatives (19, 20, 24, 25, 28–30) of the new ring system. Riboside 7 is an inhibitor of mammalian ADA (IC50 40 µM).

Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase

Rabbit skeletal muscle AMP deaminase was submitted to limited proteolysis by trypsin that converts the native 80 kDa enzyme subunit to a stable product of approx. 70 kDa, which, in contrast to the native enzyme, is not sensitive to regulation by ATP at pH 6.5. Tryptic peptide mapping indicates that proteolysis is confined to the N-terminal region of the molecule, identifying in this region of AMP deaminase a non-catalytic, 95 residue regulatory domain that stabilises the binding of ATP to a distant site in the molecule. Protein sequence analysis reveals a marked degree of divergence between rat and rabbit skeletal muscle AMP deaminases in the regions containing residues 7–12 and 51–52, giving molecular basis to the hypothesis of the existence of isoenzymes of AMP deaminase in the mature skeletal muscle of the mammals.

Elasmobranch fish ( Scyliorhinus canicula and Raja clavata) muscle AMP deaminase: Substrate specificity and effect of EDTA and adenylate energy charge

1. 1. The AMP deaminases from skeletal muscles of dogfish and skate were shown to be specific to 5′-AMP. Among several adenine nucleotide analogs, only dAMP was deaminated to an extent lower than 5%. 2. 2. Similar to vertebrates AMP deaminases, these enzymes were inhibited when incubated in the presence of EDTA solutions. 3. 3. The activity of the enzymes was regulated by adenylic energy charge variations, depending on the size of the total adenine nucleotide pool. 4. 4. The shape of the adenylate energy charge response curves of the dogfish and skate muscle AMP deaminases do not distinguish the two enzymes.

Yeast AMP deaminase. Catalytic activity in Schizosaccharomyces pombe and chromosomal location in Saccharomyces cerevisiae.

The AMP deaminase gene was mapped to chromosome XIII of Saccharomyces cerevisiae strain JM1901. The AMP deaminase gene is located near SUP5, GAL80, SUF7, and SUF22. The presence of AMP deaminase in the fission yeast Schizosaccharomyces pombe was examined by comparing DNA hybridization, protein immunoreactivity, and catalytic activity from S. cerevisiae, known to contain the protein, to S. pombe. DNA hybridization experiments using the cloned S. cerevisiae AMP deaminase gene failed to hybridize to the genomic DNA from S. pombe strain 972h-s. Protein extracts from S. pombe and S. cerevisiae were analyzed in parallel and exhibited comparable AMP deaminase activities. Analysis of reaction intermediates in cell extracts of S. pombe established that IMP is formed directly from AMP without intervening steps. The AMP deaminase of S. pombe was purified 1,100-fold to a specific catalytic activity of 67 mumol/min/mg of protein. Purified protein interacted weakly with polyclonal antibodies prepared against S. cerevisiae AMP deaminase. AMP deaminases from both S. cerevisiae and S. pombe were activated by ATP with micromolar activation constants, are inhibited by coformycin, and are specific for AMP when compared to other purine nucleosides and nucleotides. The results establish that S. pombe contains an AMP deaminase with catalytic properties similar to that from S. cerevisiae, even though the DNA sequences of the genes and the immunoreactivity of the protein from S. pombe differs considerably from the AMP deaminase of S. cerevisiae. Genetic analysis of the pathways of purine metabolism in S. pombe (Pourquié, J., and Heslot, H. (1971) Genet. Res. 18, 33-44) had indicated the absence of AMP deaminase. The presence of a regulated AMP deaminase in S. pombe supports the hypothesis that eukaryotes regulate adenine nucleotide pools by the activity of AMP deaminase.

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.

Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA.

Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.

ゴウシュウマダイ筋肉のＡＭＰデアミナーゼの精製

AMP deaminase (EC 3. 5. 4. 6) which catalyzes the deamination of AMP to IMP was solu-bilized from the skeletal muscle of the snapper Pagrus auratus with potassium phosphate buffer (pH 7.0) containing 0.1M KCl and 1mM 2-mercaptoethanol, and purified by ammonium sul-fate fractionation and three steps of column chromatography on cellulose phosphate P11, 5'AMP Sepharose 4B, and Butyl-Toyopearl 650S. The purified enzyme was shown to be homogeneous by SDS-polyacrylamide gel electrophoresis and the specific activity of the enzyme was 40.7 units/mg protein, showing 254-fold purification. The molecular weight of the native enzyme was estimated to be 3.12×105 by gel filtration and 8.2×104 by SDS-polyacrylamide slab gel electrophoresis. These results suggest that the native enzyme has a tetrameric struc-ture and that it has somewhat larger molecular weight than that of AMP deaminases from the muscle of other species.

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.

Regulatory properties of AMP deaminases from rat tissues

1. 1. Phosphoeelluse column chromatography under double gradient conditions (phosphate and KC1) revealed two forms of AMP deaminase in rat heart and brain and a single form in the liver and skeletal muscle. 2. 2. Kinetically all purified AMP deaminases were classified into two categories: those, which elute from the column at lower KCl and P i concentrations, display low S 0.5 value are only moderately affected by MgATP, MgGTP and P i; and those which elute at higher KCl and P i concentrations, display high S 0.5 values and are strongly regulated by allosteric effectors. 3. 3. Physiological significance of the occurrence of two kinetic forms of AMP deaminase in some tissues is discussed.

Comparison of kinetic and regulatory properties of high S 0.5 form of AMP deaminase from chicken and pigeon liver with AMP deaminase from rat and ox liver

1. 1. The high S 0.5 form of AMP deaminase from avian liver was shown to display a two times lower S 0.5 value than the single mammalian enzyme form. 2. 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. 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. 4. In the presence of 3 mM ATP the activity of avian enzymes was not affected by high GTP and Pi concentration, in contrast to AMP diaminase from rat liver which was strongly inhibited by GTP under the same experimental conditions. 5. 5. The differences of the regulatory properties described are discussed in terms of adjustment of avian liver AMP diaminase to a faster adenylates' catabolism and thus urate synthesis.

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 epothelium. 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 P i. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and P i. The S 0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S 0.5 decreased to 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 P i. Mg 2+ significantly altered the effects exerted by nucleotides. The S 0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and P i. When MgATP was present, AMP deamise II from rat small intestine was less susceptible to inhibition by GTP and P i. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in V max observed in the presence of MgCI 2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. Guo PP[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.

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.

Comparative study on vertebrate liver AMP deaminases

1. 1. Similar activity of AMP deaminase was found in rat, hen, turtle and flounder liver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. 2. 2. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. 3. 3. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. 4. 4. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.

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.