Kanie O, Crawley S C, Palcic M M, Hindsgaul O
Department of Chemistry, University of Alberta, Edmonton, Canada.
Carbohydr Res. 1993 Apr 23;243(1):139-64. doi: 10.1016/0008-6215(93)84087-m.
The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6' group of oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp (or Manp)-OR (5, R = (CH2)7CH3) to yield the sequence beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-alpha-D-Manp-(1--> 6)- beta-D-Glcp (or Manp)-OR. Biosynthetically, if beta-(1-->4)-galactosyltransferase acts first on 5, the product beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-be ta-D-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6' group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end D-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.
N-乙酰葡糖胺基转移酶-V（GlcNAcT-V）将UDP-GlcNAc中的GlcNAc转移至以β-D-GlcpNAc-(1→2)-α-D-Manp-(1→6)-β-D-Glcp（或Manp）-OR（5，R = (CH2)7CH3）序列结尾的寡糖的OH-6'基团上，生成β-D-GlcpNAc-(1→2)-[β-D-GlcpNAc-(1→6)]-α-D-Manp-(1→6)-β-D-Glcp（或Manp）-OR序列。在生物合成过程中，如果β-(1→4)-半乳糖基转移酶首先作用于5，产物β-D-Galp-(1→4)-β-D-GlcpNAc-(1→2)-α-D-Manp-(1→6)-β-D-Glcp（或Manp）-OR（7）即使保留了活性OH-6'基团，也不再是GlcNAcT-V的底物。本文研究了这种活性丧失的原因。化学合成了受体三糖5的六种类似物，它们都具有还原端D-葡萄糖构型。合成方案的一个关键特征是使用1,2-二氨基乙烷高效去除N-邻苯二甲酰亚胺保护基团。在这些类似物中，末端糖单元的OH-4，即正常GlcNAc结尾的三糖5中GalT进行半乳糖基化的位点，被系统地替换为OMe、F、NH2、NHAc和H，并且其构型反转成半乳糖构型。然后对所得三糖类似物与仓鼠肾中的GlcNAcT-V的相互作用进行了动力学评估。发现所有六种化合物作为GlcNAcT-V的受体或抑制剂基本上都没有活性。得出的结论是，GlcNAcT-V天然受体的半乳糖基化破坏了受体活性，不是通过引入添加糖残基的空间位阻，而是通过破坏GlcNAc残基末端OH-4与酶之间重要的氢键相互作用。因此，这个OH-4基团被指定为GlcNAcT-V的关键极性基团。
