Frick L, Yang C, Marquez V E, Wolfenden R
Department of Biochemistry, University of North Carolina, Chapel Hill 27599.
Biochemistry. 1989 Nov 28;28(24):9423-30. doi: 10.1021/bi00450a027.
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 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 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 cytidine 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 degrees 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 x 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.(ABSTRACT TRUNCATED AT 250 WORDS)
从大肠杆菌组成型突变体中纯化至同质的胞苷脱氨酶，被发现能结合竞争性抑制剂嘧啶 - 2 - 酮核糖核苷（表观 Ki = 3.6 x 10⁻⁷ M）和5 - 氟嘧啶 - 2 - 酮核糖核苷（表观 Ki = 3.5 x 10⁻⁸ M）。酶结合导致嘧啶 - 2 - 酮核糖核苷的最大吸收波长从游离状态的303 nm变为结合状态的239 nm。结合状态的值与氢氧根离子与1,3 - 二甲基 - 2 - 氧代嘧啶鎓形成的氧加合物的值相同（239 nm），但低于N - 乙酰半胱氨酸的硫醇盐阴离子与1,3 - 二甲基 - 2 - 氧代嘧啶鎓形成的硫加合物的值（259 nm）。结果表明，嘧啶 - 2 - 酮核糖核苷作为氧加合物被胞苷脱氨酶结合，可能是共价水合物3,4 - 二氢尿苷，而不是完整的形式或涉及酶的硫醇基团的加合物。在25℃的稀溶液中，根据水的解离平衡、1 - 甲基嘧啶 - 2 - 酮的质子化以及1,3 - 二甲基嘧啶鎓阳离子与氢氧根离子的结合平衡，估计从嘧啶 - 2 - 酮核糖核苷形成单一非对映异构体3,4 - 二氢尿苷的平衡常数约为4.7 x 10⁻⁶。（摘要截短于250字）
