KINETIC EVIDENCE FOR 2 ACTIVE-SITES IN BETA-D-FUCOSIDASE OF HELICELLA-ERICETORUM
KINETIC EVIDENCE FOR 2 ACTIVE-SITES IN BETA-D-FUCOSIDASE OF HELICELLA-ERICETORUM
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DOI：
10.1016/0020-711x(83)90193-3
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
CABEZAS, JA
中科院分区：
文献类型：
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作者：
CALVO, P;SANTAMARIA, MG;CABEZAS, JA
β的动力学-蜗牛H. ericetorum进行了研究。酶显示β- D-岩藻糖苷酶，β- D-葡糖苷酶和β-葡糖苷酶D-半乳糖苷酶活性，在DEAE-纤维素层析和等电聚焦（p14.35）中均为单峰，具有相同的最适pH（5.0）。用相应的对硝基苯基糖苷作为底物，D-岩藻糖苷酶活性显示最低Km、最高Vmax和最佳Vmax/Km值; β-岩藻糖苷酶获得接近的活性值。D-葡糖苷酶; β- D-半乳糖苷酶活性在该酶中低得多。所有的动力学证据表明，这种酶有2个活性位点：岩藻糖位点和半乳糖位点。β- D-岩藻糖苷酶和β- D-葡糖苷酶活性具有相似的Km、Vmax、Vmax/Km和Ki值;这些值与β-葡糖苷酶的那些非常不同。D-半乳糖苷酶活性。β- D-岩藻糖苷和β- D-葡糖苷在混合底物实验中完全竞争共同的活性位点; β- D-半乳糖苷仅与两种糖苷部分竞争。用. δ- D-葡内酯，该酶显示出双曲线混合型抑制，主要竞争β-葡内酯。D-岩藻糖苷酶和β- D-葡糖苷酶活性（具有相同的抑制亚型），并且对β-葡糖苷酶主要是非竞争性的。D-半乳糖苷酶活性（具有不同的抑制亚型）。用. δ- D-藜芦内酯对β- D-岩藻糖苷酶和β-发现了D-葡糖苷酶活性，并且在γ-葡糖苷酶活性下，D-半乳糖酸内酯，对β-半乳糖酸内酯的抑制作用更强，检测D-半乳糖苷酶活性。 该酶被某些碳水化合物激活，可能与转糖基化机制有关。
The kinetics of .beta.-D-fucosidase of the snail H. ericetorum were studied. The enzyme shows .beta.-D-fucosidase, .beta.-D-glucosidase and .beta.-D-galactosidase activities, all associated in a single peak in DEAE-cellulose chromatography and isoelectric focusing (pl 4.35), having the same optimal pH (5.0). With the corresponding p-nitrophenyl glycosides as substrates, .beta.-D-fucosidase activity shows the lowest Km, the highest Vmax and the best Vmax/Km value; close activity values were obtained for .beta.-D-glucosidase; .beta.-D-galactosidase activity is much lower in this enzyme. All the kinetic evidence suggests that this enzyme has 2 active sites: a fuco-gluco site and a galacto site. .beta.-D-fucosidase and .beta.-D-glucosidase activities have similar Km, Vmax, Vmax/Km and Ki values; these values are very different from those of .beta.-D-galactosidase activity. .beta.-D-fucosides and .beta.-D-glucosides completely compete for a common active site in mixed-substrate experiments; .beta.-D-galactosides only partially compete with both glycosides. With .delta.-D-gluconolactone, the enzyme shows a hyperbolic mixed-type inhibition, mainly competitive for .beta.-D-fucosidase and .beta.-D-glucosidase activities (with the same inhibition sub-type), and predominantly non-competitive for .beta.-D-galactosidase activity (with different inhibition sub-type). With .delta.-D-gluconolactone more inhibition of .beta.-D-fucosidase and .beta.-D-glucosidase activities was found, and with .gamma.-D-galactonolactone, more inhibition of .beta.-D-galactosidase activity was detected. The enzyme is activated by some carbohydrates, probably in relation with a transglycosylation mechanism.