PHOTOINACTIVATION STUDIES ON ADENOSINE-DEAMINASE

Reinvestigation of direct modification of histidine residues in calf i ntestine adenosine 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 b lue led to a complete loss of enzymatic activity and the presence of i nhibitors such as purin riboside, EHNA, coformycin, showed protection against methylene blue oxidation. Kinetic analysis of the inactivation by diethylpyrocarbonate, indicated that enzyme inactivation results f rom the modification of at most one essential histidine residue. Photo inactivation of adenosine deaminase from <(Aspergillus)under bar> <(Or izae)under bar> Orizae reduces the activity of the enzyme but the acce ssibility of histidine residues in the active site seems to be lower a s compared to that shown from mammalian adenosine deaminase. The resul ts obtained are in agreement with the important role played by the ''b ridge'' between an enzyme histidine residue and the 5'-OH of the ribos e mojety of substrate in the transition state stabilization in the two enzymatic forms observed. Upon ultraviolet irradiation of calf intest ine adenosine deaminase in the presence of 2,2,2-thrichloroethanol, th e trasformation of fluorescent tryptophan residues occurs with the red uced enzymatic catalytical activity and confirms the likely location o f tryptophans near the binding site of the enzyme.