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 catal ytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine dea minase was produced by dialysis in the presence of specific zinc chela tors in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult c ofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activ ity. 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 tryp tophan fluorescence of apo- or holoenzyme upon mixing with the transit ion state analog, deoxycoformycin. Excess levels of adenosine or N-6-m ethyladenosine incubated with the apoenzyme prior to the addition of m etal prevent restoration, suggesting that the cofactor adds through th e substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn 2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase acti vity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competiti ve inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (K-i greater tha n or equal to 1000 mu M) was noted for Ca2+, Fe2+, and Fe3+.