Hou Y, Vocadlo D, Withers S, Mahuran D
The Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Biochemistry. 2000 May 23;39(20):6219-27. doi: 10.1021/bi992464j.
Tay-Sachs or Sandhoff disease results from a deficiency of either the alpha- or the beta-subunits of beta-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family 20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of the three-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. The primary sequence identity between the two enzymes is only 26% and restricted to their active site regions; therefore, the validity of this model must be determined experimentally. Because human hexosaminidase cannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must be carried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity. Even small amounts of endogenous enzyme can interfere with accurate K(m) or V(max) determinations. We report the expression, purification, and characterization of a C-terminal His(6)-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments are reported confirming that the kinetic parameters of the His(6)-tag precursor are the same as the untagged precursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg(211)Lys-substituted hexosaminidase B, we reexamine the role of Arg(211) in the active site. As we previously reported, this very conservative substitution nevertheless reduces k(cat) by 500-fold. However, the removal of all endogenous activity has now allowed us to detect a 10-fold increase in K(m) that was not apparent in our previous study. That this increase in K(m) reflects a decrease in the strength of substrate binding was confirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue, i.e., a hexosaminidase affinity column. Thus, Arg(211) is involved in substrate binding, as predicted by the chitobiase model, as well as catalysis.
泰-萨克斯病或桑德霍夫病分别是由于β-己糖胺酶A的α亚基或β亚基缺乏所致。这些在进化上相关的亚基已被归类为“第20家族”糖苷酶。人类己糖胺酶的分子建模是基于第20家族的一个细菌成员——粘质沙雷氏菌壳二糖酶的三维结构进行的。这两种酶之间的一级序列同一性仅为26%，且仅限于它们的活性位点区域；因此，该模型的有效性必须通过实验来确定。由于人类己糖胺酶不能在细菌中进行功能性表达，因此必须使用所有都会产生内源性己糖胺酶活性的真核细胞表达系统来对诱变的己糖胺酶进行表征。即使是少量的内源性酶也会干扰K(m)或V(max)的准确测定。我们报告了己糖胺酶B的C末端His(6)标签前体形式的表达、纯化和表征，该前体形式不含宿主细胞内源性酶的比例为99.99%。报告的对照实验证实，His(6)标签前体的动力学参数与未标记的前体相同，而未标记的前体又与成熟同工酶相同。使用高度纯化的野生型和Arg(211)Lys取代的己糖胺酶B，我们重新审视了Arg(211)在活性位点中的作用。正如我们之前报道的，这种非常保守的取代仍然使k(cat)降低了500倍。然而，现在去除所有内源性活性后，我们检测到K(m)增加了10倍，这在我们之前的研究中并不明显。突变同工酶无法有效结合固定化底物类似物（即己糖胺酶亲和柱），证实了K(m)的这种增加反映了底物结合强度的降低。因此，正如壳二糖酶模型所预测的，Arg(211)既参与底物结合，也参与催化作用。
