Purification and characterization of N-acetylglucosamine kinase from rat liver -: Comparison with UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase
Purification and characterization of N-acetylglucosamine kinase from rat liver -: Comparison with UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase
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DOI：
10.1046/j.1432-1327.1998.2520133.x
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发表时间：
1998-02-15
期刊：
影响因子：
--
通讯作者：
Reuter, W
中科院分区：
文献类型：
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作者：
Hinderlich, S;Nöhring, S;Reuter, W
N-乙酰葡糖胺是复合碳水化合物中的主要糖，通过N-乙酰葡糖胺激酶的作用进入氨基糖代谢途径（EC 2.7.1.59）。在这项研究中，我们报告的纯化，从大鼠肝胞质溶胶中的GlcNAc激酶的均匀性，使用硫酸盐沉淀，色谱苯基琼脂糖，ATP-琼脂糖和MonoQ，最后凝胶过滤Superdex 200。其特征为39 kDa亚基的二聚体。通过肽图确定了GlcNAc激酶的约25%的氨基酸序列。GlcNAc激酶的部分ATP结合位点通过与相关己糖激酶（包括双功能酶UDP-N-乙酰葡糖胺2-差向异构酶/N-乙酰甘露糖胺激酶）的序列比较来鉴定（EC 5.1.3.14/2.7.1.60），大鼠肝脏中N-乙酰神经氨酸生物合成的关键酶。GlcNAc激酶和ManNAc激酶活性位点的Cys残基通过N-化学修饰进行表征乙基马来酰亚胺、碘乙酰胺和5，5 ′-二硫代双（2-硝基苯甲酸）。底物GlcNAc和ManNAc保护它们各自的酶免受上述巯基试剂的抑制的发现表明Cys残基位于两种酶的活性位点中或附近。使用特定的二硫醇修饰化学试剂，偏高碘酸钠，偏亚砷酸钠/2，3-二巯基丙醇和二氮烯二羧酸双-N，N '-二甲基酰胺，揭示了GlcNAc激酶和ManNAc激酶的活性位点具有至少一对邻位硫醇。UDP-GlcNAc 2-差向异构酶的化学处理没有提供在该酶的活性位点中存在半胱氨酸的证据。从N-[H-3]乙基马来酰亚胺掺入GlcNAc激酶的配体的存在和不存在下，我们估计，GlcNAc激酶的活性位点包含两个半胱氨酸残基。
N-Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase (EC 2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39-kDa subunits. About 25% of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (EC 5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver.The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N-ethylmaleimide, iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol-modifying chemical reagents, sodium meta-periodate, sodium meta-arsenite/2,3-dimercaptopropanol and diazenedicarboxylic acid bis-N,N'-dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDP-GlcNAc 2-epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N-[H-3]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.