The role of divalent cations in structure and function of murine adenosine deaminase

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn 2+ or Co 2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo‐ and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo‐ or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N 6 ‐methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca 2+ , Cd 2+ Cr 2+ , Cu + , Cu 2+ , Mn 2+ , Fe 2+ , Fe 3+ , Pb 2+ , or Mg 2+ did not restore adenosine deaminase activity to the apoenzyme. Mn 2+ , Cu 2+ , and Zn 2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd 2+ and Co 2+ were noncompetitive inhibitors. Weak inhibition (K i ≥ 1000 μM) was noted for Ca 2+ , Fe 2+ , and Fe 3+ .