Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1
Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1
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DOI：
10.1128/jb.187.2.480-487.2005
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Sugai, M
中科院分区：
文献类型：
--
作者：
Fujiwara, T;Aoki, S;Sugai, M
ALE-1是一种由葡萄球菌EPK 1产生的甘氨酰甘氨酸内肽酶。ALE-1含有7个组氨酸。使用焦碳酸二乙酯和碘乙酸进行的化学修饰研究表明，分子中的组氨酸或酪氨酸残基对生物体的葡萄球菌溶解活性很重要。所有的组氨酸残基，一个酪氨酸，和一个天冬氨酸残基的N-末端截短的ALE-1（AN-终端ALE-1）系统地改变了定点诱变，和酶的活性和金属含量的变体进行了测量。我们的研究表明His-150、His-200、His-231、His-233和Asp-154是DeltaN-term ALE-1的酶活性所必需的。除His-150和Asp-154外，所有这些氨基酸都位于11个蛋白质中的38个氨基酸的保守区域内，包括5个葡萄球菌内肽酶。电感耦合等离子体质谱分析的DeltaN-长期ALE-1显示，它包含一个原子的锌每分子。突变体Δ N-端ALE-1的锌含量的测量表明，His-150和-233对于锌结合是重要的;它们在这些变体酶中的损失与葡萄球菌溶解活性的损失一致。这些结果有力地表明ALE-1是锌金属蛋白酶的新成员。
A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (AN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.