Mycoplasma capricolum PTS Enzyme I Fragments

酶I的活性部位His上的磷酸基被转移到HPR的活性部位His残基上，有人认为酶I与HPR之间的主要相互作用是通过酶I氨基末端的α螺旋结构域进行的。用远紫外圆二色谱(CD)分离得到含有80%α螺旋的重组α螺旋结构域(残基25-145)以及从羊肚菌中分离的缺失该结构域的酶I[Ei(减去HD)]，CD估计该结构域的α螺旋含量为50%。我们发现HPR在pH 7.5和25℃时分别与α螺旋结构域和完整的酶I结合，表观结合常数分别为50,000和140,000/M，但不与缺失突变体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。然而，在沉降平衡实验中，完整的酶I和EI(Mnus HD)均为二聚体；数据分析表明，在pH 7.5和4℃或20℃时，酶I的二聚反应的对数K值分别为5.0和6.7。此外，酶I的C-末端结构域(38,027 Da，His标记由Met和6-His组成)与logK值约为10有很强的自结合。因此，酶I的N-末端结构域对酶I的二聚反应产生负面影响，从而对酶I与PEP的自磷酸化反应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端区活性部位组氨酸的磷酸化是否也影响酶I的二聚化程度，这一点还有待证实。-PEP：糖磷酸转移酶系统，磷酸转移，酶I的N-末端和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