Stereoselectivity of the Chinese hamster ovary cell sialidase: Sialoside hydrolysis with overall retention of configuration
Stereoselectivity of the Chinese hamster ovary cell sialidase: Sialoside hydrolysis with overall retention of configuration
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DOI：
10.1093/glycob/7.4.559
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发表时间：
1997-06-01
期刊：
影响因子：
4.3
通讯作者：
Warner, TG
中科院分区：
文献类型：
--
作者：
Kao, YH;Lerner, L;Warner, TG
以4-甲基伞形酮基-N-乙酰神经氨酸为底物，用质子核磁共振波谱法研究了中国仓鼠卵巢细胞可溶性唾液酸酶在昆虫Sf 9细胞中表达的重组蛋白水解的立体化学过程，并且通过监测唾液酸产物的H3轴向质子和赤道质子的立体选择性来确定酶催化作用。反应过程：在高（3U）和低（1U）浓度的酶下，唾液酸的α异头物作为初始反应产物被清楚地观察到，相应的唾液酸的β异头物在反应中出现得晚得多，由α异头物的变旋引起。使用鼠伤寒沙门氏菌LT 2唾液酸酶也进行了类似的研究，这两种酶都能切割α-连接的唾液酸糖苷底物，并保持其构型。基于对其他各种糖水解酶的观察，这些酶显示出催化的立体选择性与活性位点拓扑结构有很强的相关性，（Gebler等，J，Biol，Chem，267，12559-12561，1992），此处获得的结果表明，微生物和哺乳动物唾液酸酶具有同源活性位点结构，即使这些分子不具有显著的一级序列相似性。
The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, expressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spectroscopy, 4-Methyl umbelliferyl-N-acetyl neuraminic acid was employed as substrate, and the stereoselectivity of the enzyme catalysis was ascertained by monitoring the H3 axial and equatorial protons of the sialic acid product over the reaction course, At both high (3 U) and low concentrations (1 U) of the enzyme, the alpha anomer of the sialic acid was clearly observed as the initial reaction product, The corresponding beta anomer of sialic acid appeared much later in the reaction, arising from mutarotation of the alpha anomer, Similar studies were also carried out using the Salmonella typhimurium LT 2 sialidase, a protein of similar size and substrate specificity, Both enzymes apparently cleave the alpha linked sialoside substrate with retention of configuration, Based on the observations of a wide variety of other glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis,vith active site topology (Gebler et at, J, Biol, Chem, 267, 12559-12561, 1992), the results obtained here suggest that the microbial and mammalian sialidases have a homologous active site architecture even though the molecules do not share significant primary sequence similarities.