The mechanism of dextransucrase action. Biosynthesis of branch linkages by acceptor reactions with dextran.
The mechanism of dextransucrase action. Biosynthesis of branch linkages by acceptor reactions with dextran.
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右旋糖酐蔗糖酶的作用机制。
DOI：
10.1016/0003-9861(76)90331-3
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发表时间：
1976
影响因子：
3.9
通讯作者：
Hajime Taniguchi
中科院分区：
文献类型：
--
作者：
J. Robyt;Hajime Taniguchi
来自肠膜明串珠菌 B-512 的葡聚糖蔗糖酶催化蔗糖聚合葡聚糖。所得葡聚糖具有 95% α-1 → 6 个连接和 5% α-1 → 3 个分支连接。将纯化的葡聚糖蔗糖酶不溶解在Bio-Gel P-2珠子(BGD，Bio-Gel-葡聚糖蔗糖酶)上。通过将BGD与极低浓度的[14C]蔗糖一起孵育来标记BGD，或者首先将其装入未标记的蔗糖，然后用极低浓度的[14C]蔗糖进行标记。用缓冲液充分洗涤后， 14 C标记仍然附着在BGD上。这种标记材料先前被证明是[14C]葡聚糖，并被假定在还原端共价连接到酶的活性位点。当标记的 BGD 与低分子量非标记葡聚糖（受体葡聚糖）一起孵育时，所有 BGD 结合标记均以 [14C] 葡聚糖的形式释放，而当标记的 BGD 在相当的条件下单独在缓冲液中孵育时，基本上没有 [14C] 葡聚糖释放。通过用外葡聚糖酶水解，释放的[14C]葡聚糖显示为轻微支化的葡聚糖。释放的葡聚糖的乙酰解产生黑糖中7.3%的放射性。用硼氢化钠还原，然后酸水解，得到葡萄糖中的所有放射性，表明黑糖被专门标记在非还原性葡萄糖单元中。这些结果表明，凭借低分子量葡聚糖的作用，[14C]葡聚糖从BGD中释放出来，并且该作用形成了新的α-1→3分支连接。提出了一种支化机制，其中受体葡聚糖上的 C3-OH 充当葡聚糖基-葡聚糖蔗糖酶复合物还原端 C1 上的亲核体，从而从葡聚糖蔗糖酶中置换葡聚糖并形成 α-1 → 3 分支连接。有人认为，分支连接的生物合成不需要单独的分支酶，而是可以通过受体葡聚糖与葡聚糖基-葡聚糖蔗糖酶复合物的反应来发生。
Dextransucrase fromLeuconostoc mesenteroidesB-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [14C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [14C]sucrose. After extensive washings with buffer, the14C label remained attached to BGD. This labeled material was previously shown to be [14C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [14C]dextran whereas essentially no [14C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [14C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [14C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C3-OH on an acceptor dextran acts as a nucleophile on C1of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.