ABSTRACT
Oxazepam and lorazepam inhibit the adenosine deaminase (ADA) differently. In the case of lorazepam temperature increment causes an increase in the inhibition potency whereas higher temperature reduces the inhibitory effect of oxazepam; which proposes the overall profounder structural changes in the case of lorazepam relative to those caused by oxazepam.
Subject(s)
Adenosine Deaminase/chemistry , Adenosine Deaminase/metabolism , Enzyme Inhibitors/metabolism , Lorazepam/metabolism , Oxazepam/metabolism , Adenosine/metabolism , Adenosine Deaminase Inhibitors , Animals , Anti-Anxiety Agents/metabolism , Anticonvulsants/metabolism , Cattle , Circular Dichroism , Computer Simulation , Hypnotics and Sedatives/metabolism , Intestinal Mucosa/enzymology , Kinetics , Ligands , Models, Molecular , Protein Conformation , Spectrometry, Fluorescence , Structure-Activity Relationship , TemperatureABSTRACT
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.
Subject(s)
Acetaminophen/pharmacology , Adenosine Deaminase Inhibitors , Enzyme Inhibitors/chemistry , Acetaminophen/chemistry , Adenosine Deaminase/metabolism , Binding, Competitive , Circular Dichroism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Kinetics , Protein Conformation , Spectrometry, Fluorescence , Structure-Activity Relationship , Temperature , ThermodynamicsABSTRACT
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.
Subject(s)
Adenosine Deaminase/metabolism , Caffeine/chemistry , Enzyme Inhibitors/chemistry , Adenosine Deaminase Inhibitors , Animals , Binding, Competitive/drug effects , Calorimetry, Differential Scanning , Cattle , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Spectrophotometry, Ultraviolet , Substrate SpecificityABSTRACT
Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18000, 22000 and 21900 kJ mol(-1) respectively.
