Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis.
Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis.
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具有中性鞘磷脂酶和溶血磷脂磷脂酶 C 活性的哺乳动物酶的催化和膜靶向的结构要求。
DOI：
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发表时间：
2000
影响因子：
4.8
通讯作者：
M. Katan
中科院分区：
文献类型：
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作者：
F. Rodrigues;A. Fensome;M. Josephs;J. Evans;R. Veldman;M. Katan
与细菌中性鞘磷脂酶的序列相似性导致了推定的哺乳动物对应物的分离，随后，在许多其他真核生物中鉴定出类似的分子。基于哺乳动物酶的序列相似性和先前的表征，我们化学修饰了特定的残基，并进行了定点诱变，以确定关键的催化残基和膜定位的决定因素。组氨酸残基的修饰和底物保护实验证明活性位点内存在反应性组氨酸残基。定点突变表明两个组氨酸残基（His-136和His-272）在催化中起重要作用，这两个残基在所有序列中都是保守的。另外两个组氨酸（His-138和His-151），仅在真核生物中保守的突变，导致中性鞘磷脂酶活性降低。除鞘磷脂外，该酶还水解溶血磷脂酰胆碱。暴露于氧化环境或使用几种特定化合物修饰半胱氨酸残基也使酶失活。定点突变的8个半胱氨酸残基和凝胶位移分析表明，这些残基不参与催化反应，并建议参与的半胱氨酸的形成/断裂的二硫键，这可能是可逆的失活的氧化化合物的基础。一系列缺失突变体的细胞定位研究，表达为绿色荧光蛋白融合蛋白，表明跨膜区含有内质网定位的决定因素。
The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.