Freeman C, Hopwood J J
Biochem J. 1986 Feb 15;234(1):83-92. doi: 10.1042/bj2340083.
Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.
人氨基磺酸硫酸酯酶通过三步四柱法从肝脏中纯化至至少20000倍的同质性，该方法包括伴刀豆球蛋白A - 琼脂糖/蓝色A琼脂糖偶联步骤、Bio - Gel HT步骤，然后是CM - 琼脂糖步骤。该方法也用于从肾脏和胎盘中纯化酶。通过SDS / 聚丙烯酰胺凝胶电泳评估，肝脏、肾脏和胎盘氨基磺酸硫酸酯酶的亚基分子量为56000。通过凝胶渗透色谱法评估，来自所有三种组织来源的酶的天然蛋白分子量在Sephacryl S - 300上约为120000，在Fractogel TSK上约为100000。天然酶可能是由亚基二聚化产生的。用人肝脏氨基磺酸硫酸酯酶与多种与体内生理底物结构方面相匹配的底物（即肝素和硫酸乙酰肝素）测定动力学参数（km和kcat.）。在结构上更复杂的底物中，天然底物的糖苷配基结构的几个方面得以保留，其周转速度比单糖底物2 - 氨基葡萄糖硫酸酯快372000倍。影响底物结合和/或酶活性的糖苷配基结构是倒数第二个残基的C - 6羧基和C - 2硫酸酯基团以及倒数第三个2 - 氨基葡萄糖硫酸酯残基。在酶促攻击下，2 - 氨基葡萄糖硫酸酯的C - 4羟基参与底物与酶的结合。如果相邻单糖残基是艾杜糖或2 - 硫酸艾杜糖，非还原端2 - 氨基葡萄糖硫酸酯上C - 6硫酸酯的存在会刺激氨基磺酸键水解和底物亲和力，但如果相邻单糖残基是艾杜糖醛酸，则会强烈抑制水解。氨基磺酸硫酸酯酶是一种外切酶，因为未检测到对内部氨基磺酸键的活性。孵育pH对评估的多种底物的酶活性的影响很复杂，并且取决于底物糖苷配基结构。在受攻击的2 - 氨基葡萄糖硫酸酯残基上存在糖苷配基C - 2硫酸酯、糖苷配基C - 6羧基和C - 6硫酸酯基团会极大地影响pH响应。结构复杂的底物有两个pH最佳值。孵育温度和缓冲液离子强度显著影响pH最佳值和酶活性。Cu2 +和SO4(2 - )离子是酶活性的有效抑制剂。
