Kinetic mechanism of Staphylococcus aureus sortase SrtA
Kinetic mechanism of Staphylococcus aureus sortase SrtA
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DOI：
10.1021/bi034391g
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发表时间：
2003-09-30
期刊：
影响因子：
2.9
通讯作者：
Ellestad, G
中科院分区：
文献类型：
--
作者：
Huang, XY;Aulabaugh, A;Ellestad, G
金黄色葡萄球菌分类酶（SrtA）是一种硫醇转肽酶。该酶催化细胞壁分选反应，其中具有包含LPXTG基序的分选信号的表面蛋白在苏氨酸和甘氨酸残基之间被切割。该蛋白的苏氨酸羧基端共价连接到肽聚糖的五甘氨酸交叉桥。分选酶的转肽酶活性已在含lpetg的肽和甘油三酯之间的体外反应中得到证实。当亲核试剂不可用时，分选酶在同一位点缓慢水解LPETG肽。在本研究中，我们分析了这两类反应在分选酶催化下的稳态动力学。动力学结果完全支持乒乓机制，即在转肽化和水解过程中形成一个共同的酰基酶中间体。然而，每个反应都有一个明显的限速步骤：转肽化反应中酰基酶的形成和水解反应中同一酰基酶的水解。我们在这项研究中也证明了金黄色葡萄球菌分类酶SrtA的亲核结合位点对二甘氨酸是特异性的。虽然酶的S1‘和S2’位点都倾向于甘氨酸残基，但S1'位点对甘氨酸是完全选择性的。超过二甘氨酸的聚甘氨酸受体亲核试剂的延长不会进一步增强结合和催化作用。
Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.