Origin of Tight Binding of a Near-Perfect Transition-State Analogue by Cytidine Deaminase:Implications for Enzyme Catalysis

摘要

Cytidine deaminase(CDA)is a zinc metalloenzyme that catalyzes the hydrolytic deamination of cytldine to uridine.Zebularine(ZEES)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_Ivalue 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-hydroxide 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 transition-state 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.