Huber R E, Gaunt M T, Hurlburt K L
Arch Biochem Biophys. 1984 Oct;234(1):151-60. doi: 10.1016/0003-9861(84)90336-9.
A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase. Two classes of compounds were found to bind well to the galactosyl form of the enzyme. One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols. On the other hand, several factors seemed to control k4. Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars. Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule. In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low. Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site. More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small. Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors.
测试了大量的糖类和醇类，以观察它们在β-半乳糖苷酶的半乳糖基形式的“葡萄糖”位点上的结合能力以及反应的难易程度。发现两类化合物能很好地结合到该酶的半乳糖基形式上。一类包含结构与吡喃糖环形式的D-葡萄糖相似的糖类和醇类，另一类由相对疏水的糖类和醇类组成。另一方面，有几个因素似乎控制着k4。与相应的环状糖类相比，直链醇类的k4值较大。此外，如果受体分子末端有羟基，其反应性（k4）比羟基只在分子中间时更高。另外，如果在末端羟甲基旁边的不对称碳原子上有一个羟基，那么它必须与葡萄糖的D构型具有相同的取向；否则，k4值较低。总体而言，结果表明，在“葡萄糖”位点上的特异性主要是由结合效应而非反应性所决定的。更具体地说，这些研究表明葡萄糖是转半乳糖基化的理想分子的原因是它从酶的半乳糖基形式上解离得非常缓慢，也就是说，k-a相对较小。因此，葡萄糖会在β-半乳糖苷酶的半乳糖基形式上保留足够长的时间，以便发生转半乳糖基化，而其他受体尽管就其k4值而言具有相同的反应性（或更高的反应性），但很容易从酶的半乳糖基形式的“葡萄糖”位点上解离，因此是较差的受体。
