Stereochemistry involved in the mechanism of action of dextransucrase in the synthesis of dextran and the formation of acceptor products
Stereochemistry involved in the mechanism of action of dextransucrase in the synthesis of dextran and the formation of acceptor products
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DOI：
10.1016/0045-2068(82)90024-4
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发表时间：
1982-06
影响因子：
5.1
通讯作者：
J. Robyt;S. H. Eklund
中科院分区：
文献类型：
--
作者：
J. Robyt;S. H. Eklund
通过分子模型研究了从leuconostoc肠系膜b - 512f中合成葡聚糖和葡聚糖蔗糖酶形成受体产物的一系列事件。其机制如下：(1)活性位点的两个亲核试剂取代两个蔗糖分子中的果糖，生成两个β-葡萄糖基中间体；(2)这两个β-葡萄糖基单位一起旋转，使得每个单位的c6 -羟基相对于另一个单位c1的α-侧；(3)一个葡萄糖基单元呈船形构象，与酶的键是轴向的；(4)另一个葡萄糖基单元的c6 -羟基氧对第一个单元的c1进行亲核攻击，取代亲核酶并形成α-1,6键；(5)新的α-1,6键的旋转使转移的葡萄糖从活性位点移开。游离的亲核酶像步骤(1)一样攻击另一个蔗糖，然后重复步骤(2)-(5)，因为生长链的还原端葡萄糖基单元呈船状构象，受到新葡萄糖基单元的c6 -羟基的攻击，取代亲核酶并形成另一个α-1,6键，围绕该键发生旋转以将生长的葡聚糖链从活性位点移除。该机制的另一个特征是一对酶促质子交换基团，它们使蔗糖的糖苷氧质子化以促进裂解，然后在聚合反应中从攻击的c6羟基上去除一个质子。受体是多羟基化合物，能够亲核攻击酶结合的β-葡萄糖基或右旋糖基单位，得到α-糖苷或右旋糖苷。注意到该酶的广泛受体特异性和一些受体产物的不寻常结构，我们提出受体特异性不是由酶结合位点本身决定的，而是由受体与葡萄糖基或右旋糖基酶中间体之间形成的氢键复合物决定的。受体对β-葡萄糖基酶c1的攻击是由与聚合催化相同的质子交换基团介导的。结果表明，α-甲基-d-葡萄糖苷、d-果糖吡喃糖、异麦芽糖、麦芽糖、β-d-甘露吡喃糖、β-d-半乳糖呋喃糖、纤维素二糖、乳糖、β、β-海藻糖、α、β-海藻糖和棉子糖的受体产物的形成与葡萄糖酶中间体的特定多重氢键有关。
A proposed sequence of events in the synthesis of dextran and in the formation of acceptor products by dextransucrase fromLeuconostoc mesenteroidesB-512F has been developed with molecular models. The following mechanism is postulated: (1) two nucleophiles at the active site displace fructose from two sucrose molecules, giving two β-glucosyl intermediates; (2) these two β-glucosyl units rotate together so that the C6-hydroxyl of each is apposed to the α-side of C1of the other; (3) one glucosyl unit assumes a boat conformation in which the bond to the enzyme is axial; (4) the C6-hydroxyl oxygen of the other glucosyl unit makes a nucleophilic attack on C1of the first, displacing the enzyme nucleophile and making an α-1,6 bond; (5) rotations about the new α-1,6 linkage remove the transferred glucose from the active site. The free enzyme nucleophile attacks another sucrose as in step (1), and then steps (2)–(5) are repeated as the reducing-end glucosyl unit of the growing chain assumes the boat conformation and is attacked by the C6-hydroxyl of the new glucosyl unit, which displaces the enzyme nucleophile and forms another α-1,6 linkage, about which rotations occur to remove the growing dextran chain from the active site. An additional feature of the mechanism presented here is a pair of enzymic proton-exchange groups, which protonate the glycosidic oxygen of sucrose to facilitate cleavage, and then remove a proton from the attacking C6hydroxyl during the polymerization reaction.Acceptors are polyhydroxy compounds which are capable of nucleophilic attack on enzyme-bound β-glucosyl or dextranosyl units to give α-glucosides or dextranosides. Noting the broad acceptor specificity of the enzyme and the unusual structure of some of the acceptor products, we have proposed that acceptor specificity is determined not by an enzymic binding site per se, but by the formation of hydrogen-bonded complexes between the acceptors and the glucosyl or dextranosyl enzyme intermediates. The acceptor attack on C1of the β-glucosyl enzyme is mediated by the same proton-exchange group as that proposed for catalysis of polymerization. It is shown that specific multiple hydrogen bonding to the glucosyl-enzyme intermediate can account for the formation of the observed acceptor products from α-methyl-d-glucoside,d-fructopyranose, isomaltose, maltose, β-d-mannopyranose, β-d-galactofuranose, cellobiose, lactose, β,β-trehalose, α,β-trehalose, and raffinose.