Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase
Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase
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DOI：
10.1074/jbc.m513754200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Imanaka, T
中科院分区：
文献类型：
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作者：
Matsumi, R;Atomi, H;Imanaka, T
信号肽酶(SPPs)是信号肽从前体蛋白中释放出来后，参与信号肽初始降解的酶。与天冬氨酸肽酶的真核生物酶不同，原核生物SPPs的催化机制尚不清楚。在这项研究中，我们鉴定了来自高温考古菌科达-卡拉氏热球菌(SPPA(TK))的SPP，它们是酶活性所必需的氨基酸残基。Delta N54SppA(TK)是一种截短的蛋白，没有N-端54个残基和可能的跨膜结构域，具有很高的肽酶活性，被用作野生型蛋白。选择了16个在古细菌SPP同源序列中高度保守的残基，并用丙氨酸残基取代。突变S162A和K214A使该蛋白的多肽酶活性丧失，而所有其他突变蛋白都不同程度地表现出活性。结果表明，Ser(162)作为亲核丝氨酸，Lys(214)作为一般碱，在SPPA(TK)中形成Ser/Lys催化二聚体。动力学分析表明，Ser(184)、His(191)、Lys(209)、Asp(215)和Arg(221)支持多肽酶活性。耐人寻味的是，大量突变导致了该酶活性水平的提高。特别是，Ser(128)和Tyr(165)的突变不仅增加了SPPA(TK)的活性水平，而且扩大了底物特异性，表明这些残基的存在可能是为了防止酶在细胞中切割意外的肽/蛋白质底物。对原核生物SPP序列的详细比对有力地表明，大多数古生菌酶以及枯草芽孢杆菌的细菌酶对多肽的水解采用相同的催化机制。
Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus koda-karaensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. Delta N54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191), Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.