Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen

Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures.     

Molecular recognition of adenosine deaminase: 15N NMR studies

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-15N- and 2D-(1H-15N)- NMR using the labeled primary inhibitor [15N4]-PR. We synthesized both [15N4]-PR and an [15N4]-HDPR model, from relatively inexpensive 15N sources. The [15N4]-HDPR model was used to simulate H-bonding and possible Zn2+-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by 15N-NMR monitored pH-titrations of [15N4]-PR. Finally, we investigated the [15N4]-PR-ADA 1:1 complex by 2D-(1H-15N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn2+-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the 15N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.     

Bovine skeletal muscle adenosine deaminase: kinetic and thermodynamic studies

Adenosine deaminase from bovine skeletal muscle catalyzes the hydrolytic deamination of adenosine to inosine and ammonia via an ordered Uni-Bi mechanism, if water is not considered as a true second substrate, as deduced from the inhibition pattern products. The inhibition constants (Ki) obtained for inosine and ammonia were 316 mumol/l and 2 mol/l, respectively. The activation energy of the reaction has been calculated as 10 kcal/mol, delta H* and delta F* as 7.9 and 15.6 kcal/mol, respectively, and delta S* as -23 cal/mol/degrees K.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.     

Transition-state stabilization by adenosine deaminase: 1,6-addition of water to purine ribonucleoside, the enzyme's affinity for 6-hydroxy-1,6-dihydropurine ribonucleoside, and the effective concentration of substrate water at the active site

Positions of equilibria of highly unfavorable addition reactions, whose products are present at concentrations below the limits of detection, can be determined from equilibria of combination of anionic nucleophiles with quaternized enamines. Applied to the newly prepared 1-methylpurinium ribonucleoside cation, this method yields approximate equilibrium constants of 2 X 10(-9) M-1 for addition of water and 4 X 10(-5) M-1 for addition of N-acetylcysteine to neutral purine ribonucleoside, in dilute aqueous solution. Positions of 13C magnetic resonances and UV absorption maxima of the above complexes and comparison with those of adenosine deaminase complexes strongly suggest that purine ribonucleoside is bound by adenosine deaminase as the 1,6 covalent hydrate, not as a covalently bonded complex formed by addition of a thiol group at the active site. The favorable position of equilibrium of the hydration reaction on the enzyme, together with its extremely unfavorable position in free solution, indicates that the effective activity of substrate water at the active site is in the neighborhood of 10(10) M. The Ki value of the active diastereomer of 6-hydroxy-1,6-dihydropurine ribonucleoside is estimated as 1.6 X 10(-13) M, more than 8 orders of magnitude lower than the apparent dissociation constants of enzyme complexes with the substrate adenosine or the product inosine. The enzyme's remarkable affinity for this hydrated species, which is vanishingly rare in free solution, seems understandable in terms of the hydrate's close resemblance to a hydrated intermediate approaching the transition state in direct water attack on adenosine.     

Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.     

Influence of treatment with 3′-deoxyadenosine associated deoxycoformycin on hematological parameters and activity of adenosine deaminase in infected mice with Trypanosoma evansi

This study aimed to verify the effect of 3′-deoxyadenosine and deoxycoformycin on hematologic parameters and adenosine deaminase (ADA) activity in plasma and brain of mice infected with Trypanosoma evansi. Seventy animals were divided into seven groups, which were divided into two subgroups each for sampling on days 4 and 8 post-infection (PI). The groups were composed of three uninfected groups (A–C), namely, not-treated (A), treated with 3′-deoxyadenosine (B), and treated with deoxycoformycin (C) and four infected groups, mice with T. evansi (D–G), namely, not-treated (D), treated with 3′-deoxyadenosine (E), treated with deoxycoformycin (F), and treated with a combination 3′-deoxyadenosine and deoxycoformycin (G). Hematological parameters and ADA activity were evaluated in plasma and brain. Animals in groups B and C exhibited a reduction in the levels of plasma total protein compared group A. Animals in groups D and F showed changes in the hematological parameters. The ADA activity significantly reduced in the animals of groups C, D, F and G. Mice in the group E presented increased ADA activity in plasma. Therefore, we conclude that the treatment interferes significantly in the hematologic parameters in mice infected with T. evansi. On the other hand, when the ADA inhibitor was used we observed a significant decrease in the values of hematocrit, total erythrocytes, and hemoglobin concentration. The deoxycoformycin was able to inhibit the ADA activity of parasite thus it may be one of the mechanisms of efficacy of this treatment.     

Inhibitors of adenosine deaminase: continued studies of structure-activity relationships in analogues of coformycin

Synthesis and adenosine deaminase (ADA) inhibitory activity of two analogues of coformycin, containing the imidazo[4,5-e][1,2,4]triazepine ring system, have been reported as part of the structure-activity relationship (SAR) studies to explore the factors responsible for the extremely tight-binding characteristics of coformycins to ADA.     

Structural basis of compound recognition by adenosine deaminase

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.     

Complex of dipeptidyl peptidase II with adenosine deaminase

Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).     

1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A

Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

Kinetic, thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37 degrees C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37 degrees C and the inhibition constants are 42.8 and 96.8 microM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27 degrees C and uncompetitive at 37 degrees C with inhibition constants of 56.4 and 30.0 microM, at respectively. The binding constant and enthalpy of binding, at 27 degrees C are 45 microM, - 64.5 kJ/mol and 61 microM, - 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Kinetic,thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37°C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37°C and the inhibition constants are 42.8 and 96.8 μM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27°C and uncompetitive at 37°C with inhibition constants of 56.4 and 30.0 μM, at respectively. The binding constant and enthalpy of binding, at 27°C are 45 μM, ? 64.5 kJ/mol and 61 μM, ? 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.