title: Origin of tight binding of a near-perfect transition-state analogue by cytidine deaminase: implications for enzyme catalysis.
abstract: 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-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.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, Center of Excellence in Structural Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. hguo1@utk.edu
authors.forename: Haobo
authors.initials: H
authors.lastname: Guo
authors.forename: Niny
authors.initials: N
authors.lastname: Rao
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
mesh_terms: D003562:Cytidine / Q000031:analogs & derivatives; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011741:Pyrimidine Nucleosides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011789:Quantum Theory
chemical_list: D029968:Escherichia coli Proteins; D011741:Pyrimidine Nucleosides; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; D003564:Cytidine Deaminase