Adenosine deaminase (ADA) is a glycoprotein (34.5 kDa) consisting of a single polypeptide chain of 311 amino acids, 1,2) which catalyzes the irreversible hydrolytic deamination of adenosine and 2Ј-deoxyadenosine nucleosides to their respective inosine derivative nucleosides and ammonia with a rate enhancement of 2ϫ10 12 relative to the nonenzymatic reaction.3) Catalysis requires a Zn 2ϩ cofactor.4) The enzyme is widely distributed in vertebrates, invertebrates and mammals including humans. Aberrations in the expression and function of ADA have been implicated in several disease states such as severe combined immuno deficiency (SCID), which is characterized by impaired B-and T-cell-based immunity resulting from an inherited deficiency in ADA. 5,6) Higher level of ADA in the alimentarytract and decidual cells of the developing fetal-maternal interface put ADA among those enzymes performing unique roles related to grows rate of cells, embryo implantation, and other undetermined functions. 7,8) The inhibition of adenosine deaminase in brain would allow an accumulation of adenosine, which produces vasodilation, and increase of cerebral blood flow. Therefore the decrease of enzyme activity would potentiate the sedative actions of adenosine in interneuronal communication of the central nervous system. 9)ADA has a (a/b) barrel structure motif. The active site of ADA resides at the C-terminal end of the b barrel, in a deep oblong-shaped pocket. A pentacoordinated Zn 2ϩ cofactor is embedded in the deepest part of the pocket. The zinc ion is located deep within the substrate binding cleft and coordinated in a tetrahedral geometry to His 15, His 17, His 214, and Asp 295. A water molecule, which shares the ligand coordination site with Asp 295, is polarized by the metal giving rise to a hydroxylate ion that replaces the amine at the C6 position of adenosine through a stereo specific additionelimination mechanism.10) Mutation studies of amino acids in the proposed active site near the zinc binding site in the adenosine deaminase confirmed the essential role of these residues in catalysis. [11][12][13] ADA can hydrolyze the substituent in 6 position of a variety of substituted purine nucleosides. The enzyme' hydrolytic capabilities have been exploited to convert lipophilic 6-substituted purine nucleosides to products which show anti-HIV (human immunodeficiency virus) activity. 14,15) Understanding the interaction of ADA with its effectors at molecular level will be important in the development of the next generation of pharmaceutical agents that act as inhibitors, activator, or substrates. Isothermal titration calorimetry (ITC) gives valuable information about biomacromolecule-ligand interaction and enzyme inhibition. [16][17][18][19][20][21][22][23][24] Following our previous research on modified histidine residues 25) and inhibition study of ADA by inosine, 26) caffeine 27) and acetaminophen, 28) in this work we describe the binding properties of ADA interacted with theophylline studied by isothermal titration calorimetry us...
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.
Kinetic and thermodynamic studies were made on the effect of caffeine on the activity of adenosine deaminase in 50 mM sodium phosphate buffer, pH 7.5, using UV spectrophotometry and isothermal titration calorimetry (ITC). An uncompetitive inhibition was observed for caffeine. A graphical fitting method was used for determination of binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 350 microM by the microcalorimetry method, which agrees well with the value of 342 microM for the inhibition constant that was obtained from the spectroscopy method. Positive dependence of caffeine binding on temperature indicates a hydrophobic interaction.
Chemical modification of Adenosine Deaminase (ADA) with N-ethyl-5-phenyl isoxazoliom-3 -sulfonate (Woodward's reagent K) (WR-K) was studied using experimental and theoretical techniques. Reaction concentration ranges were 0.8-6 mM WR-K at pH 7.8 and 27 °C. It was observed that the maximum number of moles of esterified residues per mol of enzyme ( ) in this concentration ranges is 4. However, esterification of ADA does not affect the activity of ADA, suggesting that the active site residues are not esterified. Similar results were obtained when the active site was blocked with 0.1 mM erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), followed by esterification, as measured by enol ester formation using absorbance at 340 nm. A theoretical approach was employed to study the modification process using molecular dynamic simulation, MM and QM/MM minimization. A full ASA empirical model and B3LYP method were used to evaluate the relative stability of some species which may arise from the reaction of ADA with WR-K. Theoretical results have shown that five residues (Glu 244, Glu 121, Glu 337, Asp 127, Asp 338) can be possible cases for modification in reaction 1:1 between ADA and WR-K at = 1. Glu 121 was possible initially modified in this process. Besides, it is specified that atomic accessible surface area cannot be an appropriate criterion in determination of primary sites which are modified by WR-K. Ultimately, it is clarified that among effective factors in modification of enzyme surface such as atomic accessible surface, stability of modified segment and local residues changes of ADA, latter factor plays a basic role in this process from kinetics and thermodynamics point of view.
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