The role of Zn²⁺ on the structure and stability of murine adenosine deaminase

Adenosine deaminase (ADA) is a key enzyme in purine metabolism and crucial for normal immune competence. It is a 40 kDa monomeric TIM-barrel protein containing a tightly bound Zn²⁺, which is required for activity. In this study, we have investigated the role of Zn²⁺ with respect to ADA structure and stability. After removing Zn²⁺, the crystallographic structure of the protein remains highly ordered and similar to that of the holo protein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, is decreased significantly in the absence of Zn²⁺. Denaturation with urea shows the midpoint to be about 3.5 M for the apo enzyme, compared with 6.4 M for the holo enzyme. ADA contains four tryptophan residues distant from the Zn²⁺site. ¹⁹F NMR studies in the presence and absence of Zn²⁺ were carried out after incorporation of 6-¹⁹F-tryptophan. Chemical shift differences were observed for three of the four tryptophan residues, suggesting that, in contrast to the X-ray data, Zn²⁺-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn ²⁺-free protein. Real-time ¹⁹F NMR spectroscopy measuring the loss of Zn²⁺ showed that structural changes correlated with the loss of enzymatic activity.