Staphylococcus aureus sortase transpeptidase SrtA:: Insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism
Staphylococcus aureus sortase transpeptidase SrtA:: Insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism
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DOI：
10.1021/bi050141j
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发表时间：
2005-08-23
期刊：
影响因子：
2.9
通讯作者：
McCafferty, DG
中科院分区：
文献类型：
--
作者：
Frankel, BA;Kruger, RG;McCafferty, DG
在革兰氏阳性菌中，金黄色葡萄球菌转肽酶SrtA催化含LPXTG的毒力蛋白和定植相关蛋白与细胞壁肽聚糖的共价结合。最近金黄色葡萄球菌和炭疽芽孢杆菌的srtA和相关转肽酶srtB的结构特征提供了许多关于活性部位环境的细节，但也提出了关于催化和活性部位半胱氨酸硫醇激活的性质的问题。在这里，我们重新评价了srtA的动力学机制，并阐明了其催化机理的几个方面。利用稳态、稳态前、双底物动力学研究和高分辨电喷雾质谱仪，确定了重组srtA的稳态动力学参数和乒乓式水解分流动力学机制。底物Abz-LPETG-Dap(DNP)-NH_2的动力学参数k(Cat)/K-m和k(Cat)与pH呈钟形关系，k(Cat)分别为6.3+/-0.2和9.4+/-0.2，k(Cat)/K-m分别为6.2+/-0.2和9.4+/-0.2。溶剂同位素效应(SIE)显示出相反的行为，(D2O)k(CAT)为0.89+/-0.01，(D2O)(k(CAT)/K-m)为0.57+/-0.03，反映了平衡的SIE。此外，SIE测量强烈暗示，当与k(CAT)的逆线性质子库存一起考虑时，Cys 184参与了对同位素敏感的速率决定化学步骤。碘乙酰胺对SrtA的失活具有pH依赖性，表现为单次电离失活。这些研究共同为反质子化机制提供了令人信服的证据，在这种机制中，一小部分(约0.06%)的SrtA在生理pH下能够胜任催化，但却具有很高的活性，估计k(CAT)/K-m为>；10(5)M-1 S(-1)。
The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K-m and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH2 were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K-m. Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D2O)k(cat) of 0.89 +/- 0.01 and a (D2O)(k(cat)/K-m) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys 184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K-m of > 10(5) M-1 s(-1).