Carlow D, Wolfenden R
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599, USA.
Biochemistry. 1998 Aug 25;37(34):11873-8. doi: 10.1021/bi980959n.
The binding properties of substrates and competitive inhibitors of Escherichia coli cytidine deaminase are compared with those of the fragments obtained by cutting these ligands at several positions including the glycosidic bond. In contrast with the normal substrate cytidine (kcat/Km = 2.6 x 10(6) M-1 s-1), cytosine is found to serve as an extremely slow substrate (kcat/Km = 1.8 x 10(-3) M-1 s-1), despite the ability of cytosine to enter any active site that can accommodate the normal substrate cytidine. Spontaneous nonenzymatic deamination proceeds at similar rates for cytosine and cytidine at pH 7 and 25 degrees C, indicating that substituent ribose exerts little effect on the intrinsic reactivity of cytidine in solution. Dividing knon by kcat/Km, the maximal Kd value of the enzyme's complex with the altered substrate in the transition state is estimated as 6.1 x 10(-8) M for cytosine, very much higher than the value (1.2 x 10(-16) M) estimated for cytidine. The Kd value of ribofuranose, the missing substituent, is roughly 1.8 x 10(-2) M, as indicated by the Ki values of D-ribose and 1-methyl-D-ribofuranoside as competitive inhibitors. Thus, the free energy of binding of the altered substrate in the transition state is 9.5 kcal/mol more favorable for the whole molecule cytidine than for the sum of those of its parts, cytosine plus ribofuranose. As a separate molecule, however, ribose shows no detectable effect on the enzyme's activity on cytosine. Connectivity effects of similar magnitude are indicated by the equilibrium binding affinities of inhibitors. Thus, the Ki value of the transition state analogue inhibitor zebularine hydrate (1.2 x 10(-12) M) is very much lower than the combined affinities of N-ribofuranosylurea (1.6 x 10(-4) M) and allyl alcohol (0.14 M), indicating that the glycoside bond, by its presence, exerts a connectivity effect of 9.9 kcal/mol on the observed free energy of binding.
将大肠杆菌胞苷脱氨酶的底物和竞争性抑制剂的结合特性，与通过在包括糖苷键在内的几个位置切割这些配体而获得的片段的结合特性进行了比较。与正常底物胞苷（kcat/Km = 2.6×10⁶ M⁻¹ s⁻¹）相反，尽管胞嘧啶能够进入任何可容纳正常底物胞苷的活性位点，但它却是一种极其缓慢的底物（kcat/Km = 1.8×10⁻³ M⁻¹ s⁻¹）。在pH 7和25℃条件下，胞嘧啶和胞苷的自发非酶促脱氨反应速率相似，这表明取代基核糖对溶液中胞苷的固有反应性影响很小。将knon除以kcat/Km，对于胞嘧啶，酶与过渡态中改变的底物的最大Kd值估计为6.1×10⁻⁸ M，远高于对胞苷估计的值（1.2×10⁻¹⁶ M）。如D - 核糖和1 - 甲基 - D - 呋喃核糖苷作为竞争性抑制剂的Ki值所示，缺失的取代基呋喃核糖的Kd值约为1.8×10⁻² M。因此，对于整个分子胞苷而言，过渡态中改变的底物的结合自由能比对其各部分（胞嘧啶加呋喃核糖）的结合自由能之和更有利9.5千卡/摩尔。然而，作为一个单独的分子，核糖对酶对胞嘧啶的活性没有可检测到的影响。抑制剂的平衡结合亲和力表明了类似程度的连接效应。因此，过渡态类似物抑制剂水合泽布勒林（1.2×10⁻¹² M）的Ki值远低于N - 呋喃核糖基脲（1.6×10⁻⁴ M）和烯丙醇（0.14 M）的结合亲和力之和，这表明糖苷键的存在对观察到的结合自由能产生了9.9千卡/摩尔的连接效应。
