Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analog 3,4-dihydrouridine and contribution of the 4-hydroxyl group to its binding affinity

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Abstract
Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 .times. 10-7 M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 .times. 10-8 M). Enzyme binding resulted in a change of the .lambda.max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytodine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25.degree. C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 .times. 10-6, from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion. On the basis of this equilibrium constant and the apparent Ki value observed for pyrimidin-2-one ribonucleoside, the equilibrium constant for dissociation of a single inhibitory isomer of 3,4-dihydrouridine from the enzyme was estimated as 1.2 .times. 10-12 M, more than 8 orders of magnitude lower than the Km value of the substrate cytidine. Replacement of the 4-hydroxyl group of 3,4-dihydrouridine, in 3,4-dihydropyrimidin-2-one ribonucleoside (apparent Ki = 3.0 .times. 10-5 M), reduced its negative free energy of binding by 10.1 kcal/mol.