Mycoplasma capricolum PTS Enzyme I Fragments

山羊支原体 PTS 酶 I 片段

• 批准号: 6227999
• 负责人: ANN GINSBURG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227999
• 关键词: Mycoplasma; active sites; calorimetry; circular dichroism; dimer; enzyme activity; enzyme structure; histidine; intermolecular interaction; microorganism culture; phosphoproteins; phosphorylation; phosphotransferases

The phosphoryl group on the active-site His of enzyme I is transferred to the active-site His residue of Hpr, and it has been proposed that the major interaction between enzyme I and HPr occurs via the alpha- helical subdomain in the amino-terminal domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with an estimated 80 percent alpha-helices by far-UV circular dichroism (CD) spectra as well as enzyme I deficient in that domain [EI(minus HD)] from M. capricolum with an estimated 50 per cent alpha-helical content by CD were used in isothermal titration calorimetry (ITC) experiments with HPr. We found that HPr binds to the alpha-helical domain and intact enzyme I with apparent association constants of 50,000 and 140,000/M at pH 7.5 and 25 C, respectively, but HPr did not bind to the deletion mutant EI(minus HD). This is consistent with the alpha-helical domain of enzyme I being necessary for its interaction with HPr. However, intact enzyme I and EI(mnus HD) both dimerized in sedimentation equilibrium experiments; data analysis gave log K values for dimerization of 5.0 and 6.7, respectively, at pH 7.5 and either 4 or 20 C. Moreover, the C-terminal domain of enzyme I (38,027 Da with a His tag composed of Met plus 6-His) was found to strongly self- associate with a log K value of about 10. Thus, the N-terminal domain of enzyme I exerts a negative effect on enzyme I dimerization and thereby has a potential regulatory role in the autophosphorylation reaction of enzyme I with PEP, which has been thought to require the dimer structure. It remains to be proven whether phosphorylation of the active-site His in the N-terminal domain also affects the degree of dimerization of enzyme I under physiological conditions. - PEP:sugar phosphotransferase system, phosphoryl transfer, enzyme I N- and C- terminal domains, alpha helical deletion mutant of enzyme I

I酶活性位点上的磷酸基被转移到Hpr活性位点的he残基上。并且已经提出酶I和HPr之间的主要相互作用是通过酶I的氨基末端结构域的α -螺旋亚结构域发生的。分离的重组α -螺旋结构域（残基25-145）在远紫外圆二色（CD）光谱中估计有80%的α -螺旋，以及从M. capricolum中估计有50% α -螺旋含量的酶I在该结构域[EI（负HD）]中被用于等温滴定量热法（ITC）。HPr实验。我们发现，HPr在pH 7.5和25℃下分别与α -螺旋结构域和完整的酶I结合，其表观结合常数分别为50,000和140,000/M，但HPr不与缺失突变体EI（- HD）结合。这与酶I的α -螺旋结构域是其与HPr相互作用所必需的一致。然而，在沉积平衡实验中，完整的酶I和EI（不含HD）都出现了二聚体；数据分析表明，在pH 7.5和4或20℃下，二聚化的对数K值分别为5.0和6.7。此外，酶I的c端结构域（38,027 Da，其His标签由Met + 6-His组成）的对数K值约为10。因此，酶I的n端结构域对酶I二聚化产生负面影响，从而在酶I与PEP的自磷酸化反应中具有潜在的调节作用，这被认为需要二聚体结构。在生理条件下，n端活性位点His的磷酸化是否也会影响酶I的二聚化程度还有待证实。- PEP：糖磷酸转移酶系统，磷酸化转移，酶I N-和C-末端结构域，酶I的α螺旋缺失突变体