Zhang N, Peng K C, Chen L, Puett D, Pierce M
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Biol Chem. 1997 Feb 14;272(7):4225-9. doi: 10.1074/jbc.272.7.4225.
beta-1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155) catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in beta(1,6)-linkage to the alpha(1,6)-linked mannose of N-linked oligosaccharides. Circular dichroism (CD) was used to investigate the secondary structure of a recombinant, soluble form of the enzyme and its interaction with UDP-GlcNAc and an inhibitory substrate analog. The CD spectrum of the apoenzyme indicated the presence of small amounts of beta-structure and substantial amounts (>50%) of alpha-helicity. The CD spectra of solutions containing UDP-GlcNAc and different ratios of UDP-GlcNAc:enzyme were measured. Interestingly, the spectrum of each mixture could not be accounted for by simple additivity of the two individual spectra, indicating a change in environment of the chromophores and/or a conformational change of the substrate or protein concomitant with binding. Similar results were obtained with mixtures of UDP and the enzyme. Analysis of the CD difference spectra at three wavelengths yielded an estimated average Kd of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By contrast, addition of the CD spectrum of an inhibitory substrate analog of its oligosaccharide acceptor substrate and the CD spectrum of the enzyme could account for that observed of an inhibitor-enzyme mixture; moreover, addition of the inhibitor to a mixture of UDP-GlcNAc and enzyme did not alter the Kd associated with UDP-GlcNAc binding to the enzyme. These results and kinetic studies reported herein suggest an ordered reaction in which UDP-GlcNAc binds first to the enzyme, followed by the sequential binding of the trisaccharide substrate.
β-1,6-N-乙酰葡糖胺基转移酶V（EC 2.4.1.155）催化N-乙酰葡糖胺（GlcNAc）从尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）以β(1,6)键连接转移至N-连接寡糖的α(1,6)连接的甘露糖上。采用圆二色性（CD）研究该酶重组可溶形式的二级结构及其与UDP-GlcNAc和一种抑制性底物类似物的相互作用。脱辅基酶的CD光谱表明存在少量的β结构和大量（>50%）的α螺旋性。测量了含有UDP-GlcNAc以及不同UDP-GlcNAc与酶比例的溶液的CD光谱。有趣的是，各混合物的光谱无法通过两个单独光谱的简单加和来解释，这表明发色团环境发生了变化和/或底物或蛋白质在结合时发生了构象变化。UDP与酶的混合物也得到了类似结果。对三个波长处的CD差光谱进行分析，得出UDP-GlcNAc的估计平均解离常数（Kd）为4.4 mM，UDP为3.8 mM。相比之下，添加其寡糖受体底物的抑制性底物类似物的CD光谱与酶的CD光谱可解释抑制剂 - 酶混合物的光谱；此外，向UDP-GlcNAc与酶的混合物中添加抑制剂不会改变与UDP-GlcNAc结合至酶相关的Kd。本文报道的这些结果和动力学研究表明存在一种有序反应，其中UDP-GlcNAc首先与酶结合，随后是三糖底物的顺序结合。
