Matiollo Camila, Ecco Gabriela, Menegatti Angela Camila Orbem, Razzera Guilherme, Vernal Javier, Terenzi Hernán
Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil.
Biochim Biophys Acta. 2013 Jan;1834(1):191-6. doi: 10.1016/j.bbapap.2012.10.007. Epub 2012 Oct 24.
S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K(M) of nitrosylated PtpA was similar to its unmodified form, but the V(max) was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K(M) or V(max). These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.
S-亚硝基化与一氧化氮（NO）的信号转导和杀菌活性相关。我们最近描述了结核分枝杆菌蛋白酪氨酸磷酸酶A（PtpA）的S-亚硝基化，该酶在巨噬细胞内的分枝杆菌存活中起重要作用。这种翻译后修饰在单个半胱氨酸残基C53修饰后会降低酶的活性。本研究的目的是研究S-亚硝基化对PtpA动力学参数、热稳定性和结构的影响。观察到亚硝基化PtpA的K(M)与其未修饰形式相似，但V(max)显著降低。相反，用GSNO处理PtpA C53A，K(M)和V(max)均未改变。这些结果证实PtpA的S-亚硝基化特异性发生在非催化性的C53上，且这种修饰不影响底物亲和力。使用圆二色性（CD）和核磁共振（NMR）光谱技术表明，PtpA的S-亚硝基化降低了蛋白质的热稳定性，并在C53残基周围产生局部效应，这对蛋白质的稳定性和功能均产生了干扰。
