FUNCTIONAL RESIDUES AT THE ACTIVE-SITE OF BOVINE BRAIN ADENOSINE-DEAMINASE

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing gro ups with pK values of 5.4, that must be unprotonated, and 8.4, that mu st be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modif ication with (DEP). The histidine specific reagent inactivated the enz yme following a pseudo first-order kinetics with a second-order rate c onstant of 8.9 10(-3) (+/-1.8 10(-3)) M-1, min(-1). The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are burie d in the native molecule. Tryptophan residues also showed a high heter ogeneity that was increased after binding of ground- and transition-st ate analogs to the enzyme.