Patel Himatkumar V, Vyas Kavita A, Savtchenko Regina, Roseman Saul
Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
J Biol Chem. 2006 Jun 30;281(26):17570-8. doi: 10.1074/jbc.M508965200. Epub 2006 Mar 19.
Enzyme I (EI) is the first protein in the phosphotransfer sequence of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This system catalyzes sugar phosphorylation/transport and is stringently regulated. Since EI homodimer accepts the phosphoryl group from phosphoenolpyruvate (PEP), whereas the monomer does not, EI may be a major factor in controlling sugar uptake. Previous work from this and other laboratories (e.g. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg A. (2002) Biochem. 41, 906-913), indicate that K(a) is sensitive to several parameters. We report here a systematic study of K(a) determined by sedimentation equilibrium, which showed that it varied by 1000-fold, responding to virtually every parameter tested, including temperature, phosphorylation, pH (6.5 versus 7.5), ionic strength, and especially the ligands Mg(2+) and PEP. This variability may be required for a regulatory protein. Further insight was gained by analyzing EI by sedimentation velocity, by near UV CD spectroscopy, and with a nonphosphorylatable active site mutant, EI-H189Q, which behaved virtually identically to EI. The singular properties of EI are explained by a model consistent with the results reported here and in the accompanying paper (Patel, H. V., Vyas, K. A., Mattoo, R. L., Southworth, M., Perler, F. B., Comb, D., and Roseman, S. (2006) J. Biol. Chem. 281, 17579-17587). We suggest that EI and EI-H189Q each comprise a multiplicity of conformers and progressively fewer conformers as they dimerize and bind Mg(2+) and finally PEP. Mg(2+) alone induces small or no detectable changes in structure, but large conformational changes ensue with Mg(2+)/PEP. This effect is explained by a "swiveling mechanism" (similar to that suggested for pyruvate phosphate dikinase (Herzberg, O., Chen, C. C., Kapadia, G., McGuire, M., Carroll, L. J., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2652-2657)), which brings the C-terminal domain with the two bound ligands close to the active site His(189).
酶I（EI）是细菌磷酸烯醇丙酮酸：葡萄糖磷酸转移酶系统磷酸转移序列中的首个蛋白质。该系统催化糖类磷酸化/转运，并受到严格调控。由于EI同型二聚体可从磷酸烯醇丙酮酸（PEP）接受磷酰基，而单体则不能，因此EI可能是控制糖类摄取的主要因素。本实验室及其他实验室先前的研究工作（例如，迪米特罗娃，M. N.，斯切潘诺夫斯基，R. H.，鲁维诺夫，S. B.，彼得科夫斯基，A.，以及金斯伯格，A.（2002年），《生物化学》41卷，906 - 913页）表明，解离常数（K(a)）对多个参数敏感。我们在此报告一项通过沉降平衡法测定K(a)的系统研究，结果显示其变化幅度达1000倍，对几乎所有测试参数都有响应，包括温度、磷酸化、pH值（6.5对7.5）、离子强度，尤其是配体镁离子（Mg(2+)）和PEP。这种变异性对于一种调节蛋白而言可能是必要的。通过沉降速度法、近紫外圆二色光谱法以及对不可磷酸化的活性位点突变体EI - H189Q进行分析，我们获得了进一步的认识，该突变体的行为与EI几乎完全相同。EI的独特性质可由一个与本文及随附论文（帕特尔，H. V.，维亚斯，K. A.，马图，R. L.，索思沃思，M.，佩勒，F. B.）一致的模型来解释。我们认为EI和EI - H189Q各自都包含多种构象体，并且随着它们二聚化、结合Mg(2+)以及最终结合PEP，构象体数量逐渐减少。单独的Mg(2+)只会引起结构上微小的或无法检测到的变化，但Mg(2+)/PEP会引发大幅度的构象变化。这种效应可由一种“旋转机制”（类似于丙酮酸磷酸二激酶所提出的机制（赫茨伯格，O.，陈，C. C.，卡帕迪亚，G.，麦圭尔，M.，卡罗尔，L. J.，诺，S. J.）来解释，该机制使带有两个结合配体的C末端结构域靠近活性位点组氨酸（His(189)）。
