Control of glycoprotein synthesis. Kinetic mechanism, substrate specificity, and inhibition characteristics of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.
Control of glycoprotein synthesis. Kinetic mechanism, substrate specificity, and inhibition characteristics of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.
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DOI：
10.1016/s0021-9258(18)45643-8
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发表时间：
1987-04
期刊：
影响因子：
--
通讯作者：
B. Bendiak;H. Schachter
中科院分区：
文献类型：
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作者：
B. Bendiak;H. Schachter
纯化的大鼠肝脏 UDP-GlcNAc:α-D-甘露糖苷 β 1-2 N-乙酰葡糖胺基转移酶 II (Bendiak, B. 和 Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) 已进行了动力学表征，并确定了其底物特异性和抑制特性。动力学数据表明有序或很大程度上有序的顺序机制，UDP-GlcNAc 在受体之前结合。完全活性所需的最小受体结构为：（公式：参见文本）受体分子必须具有末端 Man α 1-6 残基和末端 GlcNAc β 1-2Man α 1-3 分支才能显示任何活性，但不需要还原性 GlcNAc 残基，因为在将该残基还原为 N-乙酰氨基葡萄糖醇后，酶的活性约为 50%。其他残基（GlcNAc beta 1-2Man α 1-3 臂上的 Gal beta 1-4，或 beta-Man 残基上的平分 GlcNAc beta 1-4）会消除催化活性。这些结果表明所有 N-连接复杂寡糖（二等分和不二等分的双触角、三触角和四触角）的生物合成具有严格的顺序，因为酶必须在 UDP-Gal:GlcNAc beta 1-4 半乳糖基转移酶或 N-乙酰氨基葡萄糖转移酶 III 作用之前完成作用才能形成此类结构。对核苷酸、糖、核苷酸-糖及其各自类似物的抑制研究表明，尿嘧啶5位上的氢被-CH3、溴或汞（作为硫醇盐）取代的UDP和UTP类似物是该酶良好的可逆抑制剂，而其他位点的取代通常在很大程度上降低了抑制效力。
Purified rat liver UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II (Bendiak, B., and Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) has been characterized kinetically, and its substrate specificity and inhibition characteristics have been determined. Kinetic data indicate an ordered, or largely ordered sequential mechanism, with UDP-GlcNAc binding prior to the acceptor. The minimal acceptor structure required for full activity is: (Formula: see text) The acceptor molecule must have a terminal Man alpha 1-6 residue, and a terminal GlcNAc beta 1-2Man alpha 1-3 branch to display any activity, but does not require the reducing GlcNAc residue, as the enzyme was about 50% as active after reduction of this residue to N-acetylglucosaminitol. Additional residues (Gal beta 1-4 on the GlcNAc beta 1-2Man alpha 1-3 arm, or a bisecting GlcNAc beta 1-4 on the beta-Man residue) abolish catalytic activity. These results suggest a rigid order in the biosynthesis of all N-linked complex oligosaccharides (bisected and nonbisected bi-, tri-, and tetraantennary), since the enzyme must act to completion prior to the action of either UDP-Gal:GlcNAc beta 1-4 galactosyltransferase or N-acetylglucosaminyltransferase III to make such structures. Inhibition studies with nucleotides, sugars, nucleotide-sugars, and their respective analogues revealed that analogues of UDP and UTP, in which the hydrogen at the 5 position of the uracil was substituted with -CH3, bromine, or mercury (as the mercaptide) were good reversible inhibitors of the enzyme, whereas substitution at other sites lessened the inhibitory potency, usually to a large degree.