Keresztessy Z, Kiss L, Hughes M A
Institute of Biochemistry, Lajos Kossuth University, Debrecen, Hungary.
Arch Biochem Biophys. 1994 Oct;314(1):142-52. doi: 10.1006/abbi.1994.1422.
The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was kinetically characterized in mixed substrate systems and with the transition-state analogue glucono(1-5)lactone and a series of 1-thio substrate analogues. The results indicate a common catalytic and a common sugar binding site in the enzyme for all of the investigated substrates. Kinetic parameters of the hydrolysis of linamarin and p-nitrophenyl beta-D-glucopyranoside were determined over the pH range 3.5-9.0. The pH-dependence curves gave apparent pK values of 4.5 (4.6) and 7.1 (7.3) for the free enzyme, while values of 4.5 (3.7) and 9.3 were obtained for the enzyme-substrate complexes, using either linamarin or p-nitrophenyl beta-D-glucopyranoside as the substrate. Kinetic analysis of the modification indicated that one molecule of water-soluble carbodiimide or Woodward's reagent K is required to bind to the enzyme for inactivation. The enzyme was protected against inactivation by the competitive inhibitors p-nitrothiophenyl beta-D-glucopyranoside, beta-D-glucopyranosylamine, and glucono(1-5)lactone. Spectrophotometric analysis at 340 nm showed that from the three carboxylate groups modified by Woodward's reagent K essentially one was protected by p-nitrothiophenyl beta-D-glucopyranoside. During modification Vmax decreased to 30% of that of the unmodified enzyme and Km remained unchanged. The pH dependence of inactivation showed the involvement of a group with a pK value of 4.6, indicating the modification of a carboxyl residue essential for activity. Treatment of the enzyme with the histidine-group-specific reagent diethylpyrocarbonate resulted in 80% loss of enzyme activity, in biphasic kinetics. A treatment with 0.5 M hydroxylamine at pH 7.0 regenerated 92% of the original enzyme activity. The presence of the competitive inhibitor beta-D-glucopyranosylamine protected the enzyme against inactivation, preventing the modification of one histidine residue. Statistical analysis of the residual fractional activity against the number of modified residues indicated that the modification of one histidine is responsible for 40-50% of the inactivation. The pH dependence of the inactivation gave a pK value of 7.0 for the histidine group upon which the activity depends. During modification, Vmax decreased to 30% and Km decreased to 50% of the original values.
对来自木薯（Manihot esculenta Crantz）的广谱特异性生氰β-D-葡萄糖苷酶（β-D-葡糖苷葡糖水解酶，EC 3.2.1.21）（亚麻苦苷酶）在混合底物系统中以及与过渡态类似物葡糖酸（1-5）内酯和一系列硫代底物类似物进行了动力学表征。结果表明，该酶对所有研究的底物都有一个共同的催化位点和一个共同的糖结合位点。在pH值3.5 - 9.0范围内测定了亚麻苦苷和对硝基苯基β-D-吡喃葡萄糖苷水解的动力学参数。对于游离酶，pH依赖性曲线给出的表观pK值分别为4.5（4.6）和7.1（7.3），而以亚麻苦苷或对硝基苯基β-D-吡喃葡萄糖苷为底物时，酶 - 底物复合物的pK值分别为4.5（3.7）和9.3。修饰的动力学分析表明，需要一分子水溶性碳二亚胺或伍德沃德试剂K与酶结合才能使其失活。竞争性抑制剂对硝基硫代苯基β-D-吡喃葡萄糖苷、β-D-吡喃葡萄糖胺和葡糖酸（1-5）内酯可保护该酶不被失活。在340 nm处的分光光度分析表明，伍德沃德试剂K修饰的三个羧基中，基本上有一个被对硝基硫代苯基β-D-吡喃葡萄糖苷保护。修饰过程中，Vmax降至未修饰酶的30%，而Km保持不变。失活的pH依赖性表明参与其中的基团的pK值为4.6，这表明对活性至关重要的羧基残基被修饰。用组氨酸特异性试剂焦碳酸二乙酯处理该酶导致酶活性呈双相动力学损失80%。在pH 7.0下用0.5 M羟胺处理可使92%的原始酶活性恢复。竞争性抑制剂β-D-吡喃葡萄糖胺的存在可保护该酶不被失活，防止一个组氨酸残基被修饰。对剩余分数活性与修饰残基数量进行统计分析表明，一个组氨酸的修饰导致40 - 50%的失活。失活的pH依赖性给出了对活性有影响的组氨酸基团的pK值为7.0。修饰过程中，Vmax降至原始值的30%，Km降至原始值的50%。
