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Methyl tertiary-butyl ether (MTBE) One of the gasoline additives used to increase octane and reduce greenhouse gas emissions, MTBE can enter the human blood flow through different ways including inhalation, oral and skin contact. Human carbonic anhydrase is one of the metalloenzymes that is found in almost all living organisms and has been extensively studied and many diseases are associated with carbonic anhydrase. In this study, the effect of MTBE proximity with human carbonic anhydrase II enzyme on enzyme activity was investigated by visible-ultraviolet spectroscopy and changes in enzyme Tm at different concentrations of MTBE were reported. In addition, the structural changes of the enzyme in the presence of MTBE were examined by intrinsic fluorescence spectroscopy. The results show that the enzyme activity in the presence of MTBE is inhibited by liner-complex mechanism. The results of intrinsic fluorescence spectroscopy of the enzyme show changes in the structure of the enzyme in the presence of MTBE. Also, following the binding of MTBE to the enzyme, the thermal stability of the enzyme is reduced and it becomes sensitive to temperature changes.