Glycosyltransferase activity can be modulated by small conformational changes of acceptor substrates
Glycosyltransferase activity can be modulated by small conformational changes of acceptor substrates
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DOI：
10.1021/bi034189d
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发表时间：
2003-07-22
期刊：
影响因子：
2.9
通讯作者：
Boons, GJ
中科院分区：
文献类型：
--
作者：
Galan, MC;Venot, AP;Boons, GJ
一系列在其糖苷键周围具有受限迁移率的N-乙酰基乳糖胺衍生物（化合物4-7）已被用于确定寡糖受体的构象性质的微小变化如何影响α-2，6-和α-2，3-唾液酸转移酶以及α-1，3-岩藻糖基转移酶IV和VI的糖基化的催化效率。通过在LacNAc的C-6和C-2'羟基之间引入不同长度和化学组成的系链来实现构象迁移率的限制。化合物4是高度受限的2 '，6-脱水衍生物，并且在LacNAc糖苷键处只能采用两种不寻常的构象。化合物5被亚甲基缩醛系链修饰，可以存在于更大的构象范围内;然而，Phi二面角被限制在小于30度的值，这与LacNAc的最低能量构象不完全相似。乙烯系链的6可以在LacNAc的相对大的能量平台中获得包括顺式构象A和B的构象，而通过甲基酰胺系链修饰的化合物7只能存在于B-构象中。2 ′，6-二甲氧基衍生物2用于测定5和6的C-6和C-2 ′羟基烷基化的影响，而3用于揭示7的C-6酰胺和C-2 ′烷基化的影响。测定了由α-2，6-和α-2，3-唾液酸转移酶以及α-1，3-岩藻糖基转移酶IV和VI催化的向构象受限的4-7和参比化合物1-3的转移的表观动力学参数，并且结果与它们的构象性质相关。4-6的数据表明，每种酶识别低最小能量构象的N-乙酰乳糖胺。构象性质的小变化，如在化合物5中，导致催化活性的显著损失。较大的构象变化，如在化合物4中，消除了唾液酸转移酶的所有活性，而岩藻糖基转移酶显示出一些活性，尽管非常低。化合物4和5的动力学数据清楚地表明，不同的糖基转移酶对构象变化的反应不同，岩藻糖基转移酶比唾液酸转移酶失去更少的活性。构象约束的6和7及其参比化合物2和3的相关表观动力学参数进一步支持了不同的酶反应不同的事实，并表明唾液酸转移酶和岩藻糖基转移酶识别不同构象的N-乙酰乳糖胺。总的来说，本文提供的数据表明，由例如蛋白质结构诱导的寡糖受体的小构象变化可用于调节蛋白质糖基化的模式。
A range of N-acetyllactosamine derivatives (compounds 4-7) that have restricted mobilities around their glycosidic linkages have been employed to determine how small changes in conformational properties of an oligosaccharide acceptor affect catalytic efficiencies of glycosylations by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI. Restriction of conformational mobility was achieved by introducing tethers of different length and chemical composition between the C-6 and C-2' hydroxyl of LacNAc. Compound 4 is a 2',6-anhydro derivative which is highly constrained and can adopt only two unusual conformations at the LacNAc glycosidic linkage. Compound 5 is modified by a methylene acetal tether and can exist in a larger range of conformations; however, the Phi dihedral angle is restricted to values smaller than 30degrees, which are not entirely similar to minimum energy conformations of LacNAc. The ethylene-tethered 6 can attain conformations in the relatively large energy plateau of LacNAc that include syn conformations A and B, whereas compound 7, which is modified by a methylamide tether, can only reside in the B-conformer. 2',6-Dimethoxy derivative 2 was employed to determine the effect of alkylation of the C-6 and C-2' hydroxyls of 5 and 6 whereas 3 was used to reveal the effects of the C-6 amide and C-2' alkylation of 7. The apparent kinetic parameters of transfer to the conformationally constrained 4-7 and reference compounds 1-3 catalyzed by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI were determined, and the results correlated with their conformational properties. The data for 4-6 showed that each enzyme recognizes N-acetyllactosamine in a low minimum energy conformation. A small change in conformational properties such as in compound 5 resulted in a significant loss of catalytic activity. Larger conformational changes such as in compound 4 abolished all activity of the sialyltransferases whereas the fucosyltransferases showed some activity, albeit very low. The kinetic data for compounds 4 and 5 demonstrate clearly that different glycosyltransferases respond differently to conformational changes, and the fucosyltransferases lost less activity than the sialyltransferases. Correlating apparent kinetic parameters of conformationally constrained 6 and 7 and their reference compounds 2 and 3 further supports the fact that different enzymes respond differently and indicates that sialyltransferases and fucosyltransferases recognize N-acetyllactosamine in a different conformation. Collectively, the data presented here indicate that small conformational changes of an oligosaccharide acceptor induced by, for example, the protein structure can be employed to modulate the patterns of protein glycosylation.