Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.4 (adenosine deaminase)
5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of the therapeutically important 9-beta-D-xylofuranosyladine in strongly alkaline medium with dimethyl sulphate led principally to etherification of sugar hydroxyls and, to a minor extent, to formation of products with a methylated exocyclic amino group. The various O'-methyl derivatives of xylofuranosyladenine were fractionated on a strongly basic ion exchange column, and isolated in pure form. Also isolated was 9-beta-D-xylofuranosyl-N6-methyladenine and its 2'-O-methyl derivative. The products were identified from their 1H NMR spectra, for which extensive data are tabulated. The susceptibilities of the various derivatives to calf intestinal adenosine deaminase were examined in relation to those of other adenine nucleosides; in particular, 5'-O-methylation led to total loss of substrate properties for the riboside, arabinoside and xyloside of adenine.
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PMID:Preparation of O'-METHYL derivatives of 9-beta-D-xylofuranosyladenine. 93 May 13

Cerebral energy metabolism can be measured non-invasively in unanesthetized neonatal rats with 31P NMR spectroscopy. Using this technique, serial changes in high energy phosphates were determined from the right cerebral hemispheres of 7 day postnatal rat pups during a hypoxic-ischemic insult known to produce focal brain injury. During 3 h of hypoxia-ischemia the concentration of ATP dropped to 33 +/- 8% of prehypoxic (baseline) levels, phosphocreatine (PCr)/Pi decreased from 1.5 +/- 0.51 to 0.16 +/- 0.06, while pH decreased nominally by 0.2 units. After 2.5 h of recovery in air, ATP returned to 75 +/- 10% of baseline levels, PCr/Pi rose to 1.1 +/- 0.28, and pH returned to its normal value of 7.16 +/- 0.06. This model was used to test the efficacy of the adenosine deaminase inhibitor, 2-deoxycoformycin (DCF) as a potential neuroprotective drug. The data for the drug- and saline-treated populations were analyzed by integrating ATP and Pi/PCr levels over specific time intervals, expressing it relative to baseline levels, and modeling it with cubic splines. Pretreatment with 500 micrograms/kg DCF shows a small, but statistically significant, preservation of both ATP and phosphorylation potential during hypoxia and initial recovery. Brain water content (edema) at 42 h recovery was apparently associated with both mean ATP and mean Pi/PCr in the last 2 h of hypoxia-ischemia. When ATP fell below 70% of baseline, brain edema was evident at 42 h of recovery. This methodology is suitable for extension to human infants.
NMR Biomed
PMID:31P NMR spectroscopy of perinatal hypoxic-ischemic brain damage: a model to evaluate neuroprotective drugs in immature rats. 164 72

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.
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PMID:Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase. 227 94

9-[5'-(2-Oxo-1,3,2-oxazaphosphorinan-2-yl)-beta-D-arabinosyl]adeni ne (1c) and 9-[5'-(2-oxo-1,3,2-dioxaphosphorinan-2-yl)-beta-D-arabinosyl]adeni ne (1d) were synthesized by reaction of 9-[beta-D-arabinofuranosyl]adenine with phosphoryl chloride with 1-amino-3-propanol and 1,3-propanediol, respectively. 1c consisted of a mixture of diastereomers, while 1d was enantiomerically homogeneous. The structures of these compounds were established by spectral (1H NMR, MS, UV) and elemental analyses. Both 1c and 1d were resistant to degradation by 5'-nucleotidase, alkaline phosphatase, venom phosphodiesterase, crude snake venom, adenosine deaminase, and adenylate deaminase. Neither compound was significantly biotransformed by mouse hepatic microsomal preparations in the presence of an NADPH-generating system. Compound 1c was marginally effective at prolonging the life span of mice bearing P-388 leukemia; compound 1d, however, was inactive.
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PMID:Synthesis and biological evaluation of 9-[5'-(2-oxo-1,3,2-oxazaphosphorinan-2-yl)-beta-D-arabinosyl]ade nine and 9-[5'-(2-oxo-1,3,2-dioxaphosphorinan-2-yl)-beta-D-arabinosyl]ade nine: potential neutral precursors of 9-[beta-D-arabinofuranosyl]adenine 5'-monophosphate. 241 27

Under conditions where 2'-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5'-phosphate in 15-20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of less than or equal to 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5'-phosphate in 20% overall yield. Coformycin-5'-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2'-deoxycoformycin-5'-phosphate, coformycin-5'-phosphate was a feeble substrate of snake venom 5'-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5'-AMP. With 5'-AMP analogues as substrate, the 5'-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values greater than 0.3 mM. The 5'-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki greater than 0.2 mM), but are potent inhibitors of adenylate deaminase (Ki less than or equal to 10(-9) M). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5'-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.
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PMID:Phosphorylation of coformycin and 2'-deoxycoformycin, and substrate and inhibitor properties of the nucleosides and nucleotides in several enzyme systems. 300 59

Chemical and enzymatic procedures have been employed for the preparation of various phosphorylated derivatives of the acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)adenine, an analogue of the active antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG). In combination with the previously reported 2',3'-seco nucleosides and their phosphates and cyclic phosphates (Stolarski et al., Z. Naturforsch. 41c, 758-770, 1986), this made available a broad class of acyclonucleosides and nucleotides, the acyclic moieties of which are capable of mimicking the ribose and 2'-deoxyribose rings. The solution conformations of the foregoing were determined with the aid of 1H, 13C and 31P NMR, and compared with those of DHPG and 9-(hydroxyethoxymethyl)guanine (Acyclovir, ACV). Particular attention was devoted to conformations about C-O bonds in different acyclic fragments, which demonstrated well-defined differences between 2',3'-seco derivatives on the one hand (conformational "rigidity") and derivatives with DHP and AC acyclic chains on the other (rotation about the C(1')-O(4') bond). The overall results are in good general agreement with reported crystal structures, and are compared with those obtained by quantum mechanical calculations. The conformational features of the various compounds are also discussed in relation to their substrate and/or inhibitor properties in a number of enzyme systems, including adenosine deaminase, phosphodiesterases, nuclease P1,3'-nucleotidase and herpes virus type 1 thymidine kinase.
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PMID:Solution conformations of some acyclo nucleoside and nucleotide analogues of antiviral acyclonucleosides, and their substrate/inhibitor properties in several enzyme systems. 338 56

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.
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PMID:Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study. 344 68

(-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace 125I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI = 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. [125I]HPIA prepared using carrier-free 125I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of [125I]HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM (K1 = 2.55 X 10(7) M-1 min-1; K-1 = 0.024 min-1) in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. We conclude that [125I]HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)[125I]HPIA and (+)[125I]HPIA would result in the generation of nonlinear Scatchard plots.
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PMID:Purification and characterization of (-)[125I]hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding. 609 94

The syn in equilibrium anti equilibrium conformation about the glycosidic bond of purine nucleosides and 5'-nucleotides in different solvent systems has been investigated by means of 1H NMR spectroscopy. Quantitative values for the conformer populations were improved, relative to previous results, by a detailed study of, and a resultant derived correction for, the influence of the sugar exocyclic group conformation on the chemical shifts of the sugar ring protons. This was achieved with the aid of nucleosides and nucleotides fixed in the conformations gauche-trans [derivatives of 8,5'-(R)-cyclo] and trans-gauche [derivatives of 8,5'-(S)-cyclo]. The results of 13C NMR confirmed those obtained by 1H NMR. The measured values of the vicinal coupling constants between H-1' and the C-8 and C-4 carbons were employed to evaluate approximately the glycosidic angles chi of the nucleosides in the conformations syn and anti. A critical examination is made of the applicability of relaxation methods, involving analysis of spin-lattice relaxation time of protons (T1) and the Overhauser effect, to determine the conformation of the base about the glycosidic bond; interpretations are provided for the lack of agreement between these methods and those based on chemical shifts in the present study. The foregoing resuls are also applied to an examination of the effect of the conformation of the base about the glycosidic bond on the enzymatic reactions catalyzed by 5'-nucleotidase and adenosine deaminase.
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PMID:NMR studies in the syn-anti dynamic equilibrium in purine nucleosides and nucleotides. 740 42

Mouse adenosine deaminase (ADA) contains an active site glutamate residue at position-217 that is highly conserved in other adenosine and AMP deaminases. Previous research has suggested that proton donation to N-1 of the adenosine ring occurs prior to catalysis and supports the mechanism as proceeding via formation of a tetrahedral intermediate at C-6 of adenosine. The proposed catalytic mechanism of ADA based on the recent elucidations of the crystal structure of this enzyme with transition- and ground-state analogs hypothesized that Glu217 was involved in this proton donation step [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284; Wilson, D. K., & Quiocho, F. A. (1993) Biochemistry 32, 1689-1693]. Site-directed mutagenesis of the equivalent glutamate in human ADA resulted in a dramatic loss of enzyme activity [Bhaumik, D., Medin, J., Gathy, K., & Coleman, M. (1993) J. Biol. Chem. 268, 5464-5470]. To further study the importance of this residue, site-directed mutagenesis was used to create mouse ADA mutants. Glu217 was mutated to Asp, Gly, Gln, and Ser, and all mutants were successfully expressed and purified. Circular dichroism and zinc analysis showed no significant changes in secondary structure or zinc content, respectively, compared to the native protein. The mutants showed only a slight variation in Km but dramatically reduced kcat, less than 0.2% of wild-type activity. UV difference and 13C NMR spectra conclusively demonstrated the failure of any of these mutants to hydrate purine riboside, a reaction carried out by the wild-type enzyme that results in formation of an enzyme-inhibitor complex. Surprisingly, Ki values for binding of the inhibitor to the mutants and to wild-type protein are similar, irrespective of whether the inhibitor is hydrated upon binding. These data confirm the importance of Glu217 in catalysis as suggested by the crystal structure of mouse ADA.
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PMID:Site-directed mutagenesis of active site glutamate-217 in mouse adenosine deaminase. 863 99


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