The temperature dependence of the activity and structure of the enzyme carbonic anhydrase was studied. The Arrhenius plot shows a jump which is seen usually in proteins with more than one subunit or with one subunit but more than one domain. Since carbonic anhydrase has only one subunit with one domain, the fine conformational changes of the protein motifs could only be detected through circular dichroism polarimetry. It seems that the jump in Arrhenius plot is a result of some slight structural changes in the secondary and tertiary structures of the enzyme.
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 μM by the microcalorimetry method, which agrees well with the value of 342 μM for the inhibition constant that was obtained from the spectroscopy method. Positive dependence of caffeine binding on temperature indicates a hydrophobic interaction.
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