OICC Press Iranian Journal of Catalysis 2345-4865 6 5 2024 02 03 Caffeine effect on adenosine deaminase catalysis: A new look at the effect of caffeine on adenosine deaminase activity EN Roya Mahinpour Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, Iran. Majid Ghasemi Department of Organic Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran. Zahra Moosavi-Nejad Departmentof Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran. Zohreh Zahraie Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, Iran. Journal Article 2024 02 03 The effect of physiological concentrations of caffeine (purified from Persian tea) on adenosine deaminase (ADA) activity at physiological and pathological concentrations of adenosine (as substrate) in 50 mM Tris-HCl buffer (pH 7.3) at 37°C was investigated, using UV-VIS spectroscopy. ADA exhibited a bi-phasic activity behavior and both phases showed positive cooperativities indicating adenosine has at least two regulatory sites on ADA molecule. We proposed a mechanism for the inhibitory effect of caffeine on ADA catalytic activity by analyzing ADA kinetic behavior at various physiological concentrations of caffeine (10, 30, 70 mM): Caffeine dominantly resulted in decreased catalytic activity of ADA via reducing the positive homotropic effect of adenosine on ADA activity. Surprisingly, it also showed a very weak but significant activatory effect on ADA activity in a concentration-dependent manner. To the best of our knowledge, this is the first report on the dual effect of caffeine on ADA activity.