Photoinactivation studies on adenosine deaminase

AbstractReinvestigation of direct modification of histidine residues in calf intestine adenosin deaminase resulted in loss of the activity towards substrates as adenosine, 2′‐deoxyadenosine and 6‐hydroxyl‐aminopurin riboside. Photo‐oxidation of the enzyme in the presence of methylene blue led to a complete loss of enzymatic activity and the presence of inhibitors such as purin riboside, EHNA, coformycin, showed protection against methylene blue oxidation. Kinetic analysis of the inactivation by diethylpyrocarbonate, indicated that enzyme inactivation results from the modification of at most one essential histidine residue. Photoinactivation of adenosine deaminase from Aspergillus Orizae reduces the activity of the enzyme but the accessibility of histidine residues in the active site seems to be lower as compared to that shown from mammalian adenosine deaminase. The results obtained are in agreement with the important role played by the “bridge” between an enzyme histidine residue and the 5′‐OH of the ribose mojety of substrate in the transition state stabilization in the two enzymatic forms observed. Upon ultraviolet irradiation of calf intestine adenosine deaminase in the presence of 2,2,2‐thrichloroethanol, the trasformation of fluorescent tryptophan residues occurs with the reduced enzymatic catalytical activity and confirms the likely location of tryptophans near the binding site of the enzyme.