Cytidine deaminase (CDA) is a zinc metalloenzyme that catalyzes the hydrolytic deamination of cytidine to uridine. Zebularine (ZEB) binds to CDA, and the binding process leads to a near-perfect transition-state analogue (TSA) inhibitor at the active site with an estimated K-i value of 1.2 x 10(-12) M. The interaction of CDA with the TSA inhibitor has become a paradigm for studying the tight TSA binding by enzymes. The formation of the TSA is catalyzed by CDA by a mechanism that is similar to the formation of the tetrahedral intermediate during the CDA-catalyzed reaction (i.e., through the nucleophilic attack of a Zn-hydroxide group on C-4). It is believed that the TSA formed at the active site is zebularine 3,4-hydrate. In this paper, it is shown from QM/MM molecular dynamics and free energy simulations that zebularine 3,4-hydrate may in fact be unstable in the enzyme and that a proton transfer from the Zn-hydroxide group to Glu-104 during the nucleophilic attack could be responsible for the very high affinity. The nucleophilic attack by the Zn-hydroxicle on C-4 is found to be concerted with two proton transfers. Such concerted process allows the TSA, an alkoxide-like inhibitor, to be stabilized through a mechanism that is similar to the transitionstate stabilization in the general acid-base catalysis. It is suggested that the proton transfer from the Zn-hydroxide to Glu-104, which is required to generate the general acid for protonating the leaving ammonia, may play an important role in lowering the activation barrier during the catalysis.