Chen C C, Guo W J, Isselbacher K J
Department of Medicine, Harvard Medical School, Boston, MA.
Biochem J. 1987 Nov 1;247(3):715-24. doi: 10.1042/bj2470715.
Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed.
大鼠肠道海藻糖酶经增溶、纯化后重构成蛋白脂质体。以辛基葡糖苷作为增溶去污剂，纯化后的蛋白在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上呈现为一条单一的条带，表观分子量为67 kDa。动力学研究表明，该酶的活性位点在功能上可分为两个相邻区域，即结合位点（pKa为4.8）和催化位点（pKa为7.2）。其他研究结果表明，催化位点含有一个功能性硫醇基团，该基团对N-乙基马来酰亚胺、Hg2+和碘乙酸的抑制作用敏感。底物保护和硫醇基团的碘乙酸标记表明，只有67 kDa的蛋白质被标记。此外，蔗糖和根皮苷可保护硫醇基团，但类似Tris的抑制剂则不能。对类似Tris的抑制剂进行结构抑制分析、Tris抑制的pH效应以及Tris对1-(3-二甲基氨基丙基)-3-乙基碳二亚胺失活的保护作用，有助于对一个含有用于Tris结合的羧基的单独位点进行表征和定位，该位点也可能是结合区域。基于这些发现，提出了海藻糖酶活性位点的一种可能结构。
