Kruger Ryan G, Dostal Patrick, McCafferty Dewey G
Johnson Research Foundation and the Department of Biochemistry and Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA.
Anal Biochem. 2004 Mar 1;326(1):42-8. doi: 10.1016/j.ab.2003.10.023.
The SrtA isoform of the Staphylococcus aureus sortase transpeptidase is responsible for the covalent attachment of virulence- and colonization-associated proteins to the bacterial peptidoglycan. Sortase utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-d-isoGlu-Lys(-Gly5)-d-Ala-d-Ala (branched Lipid II) and secreted proteins containing a highly conserved LPXTG sequence near their C termini. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we show that reported fluorescence quenching activity assays for SrtA are subject to marked fluorescence inner filter effect quenching, resulting in prematurely hyperbolic velocity versus substrate profiles and underestimates of the true kinetic parameters kcat and Km. We therefore devised a discontinuous high-performance liquid chromatography (HPLC)-based assay to monitor the SrtA reaction employing the same substrates used in the fluorescence quenching assay: Gly5 and Abz-LPETG-Dap(Dnp)-NH2. Fluorescence or UV detection using these substrates facilitates separate analysis of both the acylation and the transpeptidation steps of the reaction. Because HPLC was performed using fast-flow analytical columns (<8min/run), high-throughput applications of this assay for analysis of SrtA substrate specificity, kinetic mechanism, and inhibition are now feasible. Kinetic analysis using the HPLC assay revealed that the kinetic parameters for SrtA with Abz-LPETG-Dap(Dnp)-NH2 are 5.5mM for Km and 0.27s-1 for kcat. The Km for Gly5 was determined to be 140microM. These values represent a 300-fold increase in Km for the LPXTG substrate and a 12,000-fold increase in kcat over literature-reported values, suggesting that SrtA is more a robust enzyme than previous analyses indicated.
金黄色葡萄球菌分选酶转肽酶的SrtA同工型负责将与毒力和定植相关的蛋白质共价连接到细菌肽聚糖上。分选酶利用两种底物，十一异戊烯焦磷酸基-MurNAc（GlcNAc）-Ala-d-异谷氨酰胺-Lys（-Gly5）-d-丙氨酸-d-丙氨酸（分支脂II）和在其C末端附近含有高度保守的LPXTG序列的分泌蛋白。SrtA同时切割含LPXTG蛋白的Thr-Gly键，并与分支脂II的Gly5部分的亲核氨基形成新的酰胺键，将蛋白质锚定到这个关键中间体上，该中间体随后聚合成肽聚糖。在这里，我们表明，报道的SrtA荧光猝灭活性测定受到显著的荧光内滤效应猝灭影响，导致速度与底物曲线过早呈双曲线，并低估了真实的动力学参数kcat和Km。因此，我们设计了一种基于不连续高效液相色谱（HPLC）的测定方法，以监测使用荧光猝灭测定中相同底物的SrtA反应：Gly5和Abz-LPETG-Dap（Dnp）-NH2。使用这些底物的荧光或紫外检测有助于对反应的酰化和转肽步骤进行单独分析。由于HPLC是使用快速流动分析柱进行的（每次运行<8分钟），因此该测定法用于分析SrtA底物特异性、动力学机制和抑制作用的高通量应用现在是可行的。使用HPLC测定法进行的动力学分析表明，SrtA与Abz-LPETG-Dap（Dnp）-NH2的动力学参数为Km为5.5mM，kcat为0.27s-1。Gly5的Km测定为140μM。这些值表示LPXTG底物的Km比文献报道的值增加了300倍，kcat增加了12000倍，这表明SrtA是一种比以前分析表明的更强健的酶。
