Mycoplasma capricolum PTS Enzyme I Fragments

酶I的活性位点His上的磷酰基转移到Hpr的活性位点His残基上，并且已经提出酶I和Hpr之间的主要相互作用通过酶I的氨基末端结构域中的α-螺旋亚结构域发生。分离的重组α-螺旋结构域（残基25-145）具有通过远紫外圆二色性（CD）光谱估计的80%的α-螺旋以及在该结构域中缺乏的酶I [EI（减去HD）]。通过CD估计α-螺旋含量为50%的山羊草用于HPr的等温滴定量热法（ITC）实验。我们发现HPr在pH7.5和25 ℃下分别以50，000和140，000/M的表观缔合常数与α-螺旋结构域和完整的酶I结合，但HPr不与缺失突变体EI（减去HD）结合。这与酶I的α螺旋结构域对于其与HPr的相互作用是必需的一致的。然而，完整的酶I和EI（mnus HD）在沉降平衡实验中都发生了二聚化;数据分析表明，在pH7.5和4或20 ℃下，二聚化的log K值分别为5.0和6.7.此外，发现酶I的C-末端结构域（38，027 Da，具有由Met加6-His组成的His标签）与约10的log K值强烈自缔合。因此，酶I的N-末端结构域对酶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。它仍然有待证明是否磷酸化的活性位点的组氨酸在N-末端结构域也影响酶I在生理条件下的二聚化的程度。- PEP：糖磷酸转移酶系统，磷酰基转移，酶I N-和C-末端结构域，酶I的α螺旋缺失突变体

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