HUMAN-LIVER SULFAMATE SULFOHYDROLASE - DETERMINATIONS OF NATIVE PROTEIN AND SUBUNIT MR VALUES AND INFLUENCE OF SUBSTRATE AGYLCONE STRUCTURE ON CATALYTIC PROPERTIES
HUMAN-LIVER SULFAMATE SULFOHYDROLASE - DETERMINATIONS OF NATIVE PROTEIN AND SUBUNIT MR VALUES AND INFLUENCE OF SUBSTRATE AGYLCONE STRUCTURE ON CATALYTIC PROPERTIES
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DOI：
10.1042/bj2340083
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发表时间：
1986-02-15
影响因子：
4.1
通讯作者：
HOPWOOD, JJ
中科院分区：
文献类型：
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作者：
FREEMAN, C;HOPWOOD, JJ
1.采用刀豆素A-琼脂糖凝胶/Blue A琼脂糖偶联、Bio-Gel-HT、CM-琼脂糖凝胶三步四柱法，从肝脏中纯化了至少20,000倍纯度的人氨基磺酸水解酶。该方法还用于从肾脏和胎盘中提纯酶。2.用SDS/聚丙烯酰胺凝胶电泳法测定肝、肾和胎盘的磺酸盐水解酶的亚基为56,000。三种组织来源的酶的天然蛋白质经凝胶渗透层析法鉴定为约为MR。120,000人服用Sephacryl S-300,100,000人服用FractoGel TSK。这种天然酶很可能是亚基二聚化的结果。3.采用与体内生理底物的结构相匹配的多种底物，即肝素和硫酸肝素，测定了人肝氨基磺酸水解酶的动力学参数(Km和kcat)。结构更复杂的底物，其中天然底物的苷元结构的几个方面保持不变，翻转速度比单糖底物2-磺胺氨基葡萄糖快372,000倍。影响底物结合和/或酶活性的苷元结构是倒数第二个残基C-6羧基和C-2硫酸酯基团，以及倒数第二个2-氨基氨基葡萄糖残基。2-磺胺葡萄糖在酶作用下的C-4羟基参与底物与酶的结合。如果相邻的单糖残基是二糖或二硫代糖，则非还原末端2-氨基葡糖胺上的C-6硫酸酯会刺激磺酸键的水解和底物亲和力，但如果相邻的单糖残基是艾杜糖酸，则C-6硫酸酯会强烈地抑制水解。磺酸盐硫解酶是一种外切酶，因为没有检测到朝向内部磺酸键的活性。4.不同底物条件下，培养pH对酶活性的影响是复杂的，与底物苷元结构有关。糖苷酮C-2硫酸酯和糖苷酮C-6羧基和C-6硫酸酯基团存在于2-氨基氨基葡萄糖残基上，对pH响应有较大影响。结构复杂的底物有两个最适pH。培养温度和缓冲液离子强度对最适pH和酶活力有显著影响。Cu2+和SO42-离子是酶活性的有效抑制剂。
1. 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. 2. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polyacrylamide-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. 3. 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 372,000 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-sulphaminoglucamine 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. 4. 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 SO42- ions are potent inhibitors of enzyme activity.