Flight of a Cytidine Deaminase Complex with an Imperfect Transition State Analogue Inhibitor: Mass Spectrometric Evidence for the Presence of a Trapped Water Molecule

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (Kd ∼ 10–12 M), capturing all but ∼5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (Ktx ∼ 10–16 M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme–inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364–11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E2Zn2W2 (dimeric Zn-CDA with two water molecules), E2Zn2W2Fz, and E2Zn2W2Fz2, where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E2Zn2 is the only dimeric species detected, with no additional water molecules. Experiments conducted in H218O indicate that the added mass W represents a trapped water molecule rathe...