Inhibition of Escherichia coli cytidine deaminase by a phosphapyrimidine nucleoside.
Inhibition of Escherichia coli cytidine deaminase by a phosphapyrimidine nucleoside.
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发表时间：
1984-11
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通讯作者：
G. Ashley;P. Bartlett
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作者：
G. Ashley;P. Bartlett
大肠杆菌胞苷脱氨酶和磷酸嘧啶核苷1之间的相互作用的性质进行了动力学和荧光定量研究。化合物1被设计为过渡态类似物，并且是胞苷脱氨酶的有效、缓慢结合的抑制剂（阿什利，G. W.，和Bartlett，P. A.（1982）Biochem. Biophys.通信资源108，1467-1474）。我们目前的证据表明，1的结合是可逆的，没有共价键之间的酶和1。在pH 6时，从E X I复合物的解离中恢复酶活性的速率强烈依赖于E X I的浓度，表明抑制剂可逆地解离。紫外差光谱表明，1的发色团是不变的结合酶，从而消除了可逆的，共价修饰的酶的可能性。对于1的活性β-端基异构体与胞苷脱氨酶的结合，在pH 6下测定以下动力学参数：kon = 8300 M-1 S-1，koff = 7.8 × 10（-6）S-1，Ki = 0.9 nM。我们还能够观察和表征E.大肠杆菌胞苷脱氨酶，3.这种相互作用涉及到最初形成一个松散的复合物（KD = 1.2 μ M），然后异构化在一个缓慢的步骤，得到一个更紧密结合的复合物（Ki = 0.24 μ M）与正向和反向速率常数kf = 3.81 min-1和kr = 0.95 min-1，分别。
The nature of the interaction between Escherichia coli cytidine deaminase and the phosphapyrimidine nucleoside 1 has been studied kinetically and spectrophotometrically. Compound 1 was designed as a transition-state analog, and is a potent, slow-binding inhibitor of cytidine deaminase (Ashley, G. W., and Bartlett, P. A. (1982) Biochem. Biophys. Res. Commun. 108, 1467-1474). We present evidence that the binding of 1 is reversible, with no covalent linkage between the enzyme and 1. At pH 6, the rate of recovery of enzyme activity from dissociation of the E X I complex is strongly dependent on the concentration of E X I, indicating that the inhibitor dissociates reversibly. UV difference spectroscopy reveals that the chromophore of 1 is unaltered on binding to the enzyme, thus eliminating the possibility of reversible, covalent modification of the enzyme. For the binding of the active beta-anomers of 1 to cytidine deaminase, the following kinetic parameters were determined at pH 6: kon = 8300 M-1 S-1, koff = 7.8 X 10(-6) S-1, Ki = 0.9 nM. We were also able to observe and characterize time-dependent inhibition of E. coli cytidine deaminase by tetrahydrouridine, 3. This interaction involves involves initial formation of a loose complex (KD = 1.2 microM), followed by isomerization in a slow step to give a more tightly bound complex (Ki = 0.24 microM) with forward and reverse rate constants kf = 3.81 min-1 and kr = 0.95 min-1, respectively.