Tse R, Wu Y J, Vavougios G, Hou Y, Hinek A, Mahuran D J
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Biochemistry. 1996 Aug 20;35(33):10894-903. doi: 10.1021/bi960503a.
There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.
人β-己糖胺酶有三种同工酶，它们由α亚基和/或β亚基的所有可能二聚体组合构成；即A（αβ）、B（ββ）和S（αα）。两个亚基的氨基酸序列有60%相同。两条链之间的同源性在中间部分>羧基末端部分>>氨基末端部分有所不同。虽然二聚化是活性所必需的，但每个亚基都有自己的活性位点，并且在底物特异性和热稳定性方面存在差异。己糖胺酶A中β亚基的存在也会影响α亚基的底物特异性；例如，在体内只有A异二聚体能够水解GM2神经节苷脂。在本报告中，我们通过在相邻对齐残基处连接的α/β融合蛋白的细胞表达来定位两个亚基中的功能区域。首先，通过用相应的α片段替换β链中保守性最差的氨基末端部分，构建了一个嵌合α/β链。该蛋白的生化特性与己糖胺酶B几乎相同。因此，亚基中差异最大的区域并非其不同生化特性的原因。构建了第二个融合蛋白，它还包含α链中同源性更高的中间部分。该蛋白表现出含有α亚基（A和S）的同工酶所特有的底物特异性。我们得出结论，负责α亚基结合带负电荷底物能力的区域位于α132 - 283残基内。有趣的是，来自β链的剩余羧基末端部分，即β316 - 556，足以使这种嵌合体以异二聚体己糖胺酶A 10%的比活性水解GM2神经节苷脂。因此，每个亚基的羧基末端部分可能参与亚基 - 亚基相互作用。
