Casabon Israël, Couture Manon, Vaillancourt Katy, Vadeboncoeur Christian
Groupe de recherche en écologie buccale (GREB), Faculté de Médecine Dentaire, and Département de Biochimie et de Microbiologie, Université Laval, Quebec City, Quebec, Canada.
Biochemistry. 2009 Nov 17;48(45):10765-74. doi: 10.1021/bi901512b.
HPr is a central protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In streptococci, HPr can be phosphorylated at His(15) at the expense of PEP by enzyme I (EI) of the PTS, producing HPr(His approximately P). HPr can also be phosphorylated at Ser(46) by the ATP-dependent HPr(Ser) kinase/phosphorylase (HprK/P), producing HPr(Ser-P). Lastly, HPr can be phosphorylated on both residues, producing HPr(Ser-P)(His approximately P) (HPr-P2). We report here a study on the phosphorylation of Streptococcus salivarius HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) by HprK/P to assess the involvement of HprK/P in the synthesis of HPr-P2 in streptococcal cells. We first developed a spectrophotometric method for measuring HprK/P kinase activity. Using this assay, we found that the K(m) of HprK/P for HPr at pH 7.4 and 37 degrees C was approximately 110 muM, with a specificity constant (k(cat)/K(m)) of 1.7 x 10(4) M(-1) s(-1). The specificity constants for HPr(H15D) and HPr(H15E) were approximately 13 times lower. Kinetic studies conducted under conditions where HPr(His approximately P) was stable (i.e., pH 8.6 and 15 degrees C) showed that HPr(His approximately P) was a poorer substrate for HprK/P than HPr(H15D), the k(cat)/K(m) for HPr(H15D) and HPr(His approximately P) being approximately 9 and 26 times lower than that for HPr, respectively. Our results suggested that (i) the inefficiency of the phosphorylation of HPr(His approximately P) by HprK/P results from the presence of a negative charge at position 15 as well as from other structural elements and (ii) the contribution of streptococcal HprK/P to the synthesis of HPr-P2 in vivo is marginal.
HPr是磷酸烯醇丙酮酸：糖磷酸转移酶运输系统（PTS）的核心蛋白。在链球菌中，PTS的酶I（EI）可以消耗磷酸烯醇丙酮酸（PEP）将HPr的第15位组氨酸磷酸化，生成HPr（HisP）。HPr也可以被ATP依赖性的HPr（Ser）激酶/磷酸酶（HprK/P）将第46位丝氨酸磷酸化，生成HPr（Ser-P）。最后，HPr的这两个残基都可以被磷酸化，生成HPr（Ser-P）（HisP）（HPr-P2）。我们在此报告一项关于唾液链球菌HPr、HPr（H15D）、HPr（H15E）和HPr（HisP）被HprK/P磷酸化的研究，以评估HprK/P在链球菌细胞中HPr-P2合成中的作用。我们首先开发了一种用于测量HprK/P激酶活性的分光光度法。使用该测定法，我们发现HprK/P在pH 7.4和37℃下对HPr的K(m)约为110μM，特异性常数（k(cat)/K(m)）为1.7×10⁴ M⁻¹ s⁻¹。HPr（H15D）和HPr（H15E）的特异性常数约低13倍。在HPr（HisP）稳定的条件下（即pH 8.6和15℃）进行的动力学研究表明，HPr（HisP）作为HprK/P的底物比HPr（H15D）差，HPr（H15D）和HPr（HisP）的k(cat)/K(m)分别比HPr低约9倍和26倍。我们的结果表明：（i）HprK/P对HPr（His~P）磷酸化效率低下是由于第15位存在负电荷以及其他结构元件；（ii）链球菌HprK/P在体内对HPr-P2合成的贡献微乎其微。
