Nichtl A, Buchner J, Jaenicke R, Rudolph R, Scheibel T
Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätstr. 31, Regensburg, 93040, Germany.
J Mol Biol. 1998 Oct 9;282(5):1083-91. doi: 10.1006/jmbi.1998.2075.
beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present. Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme. Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates. The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.
来自大肠杆菌的β-D-半乳糖苷酶是目前已知的最大的四聚体酶之一。尽管其生理重要性、合成调控、酶学性质和结构已得到充分确立，但关于该酶的稳定性和折叠途径却知之甚少。在此我们表明，化学变性的β-半乳糖苷酶的整体折叠机制包括三个阶段：(i) 二级结构元件的形成；(ii) 折叠成亚结构域和结构化单体；(iii) 通过二聚体中间体缔合形成天然四级结构。第一个限速步骤是结构化单体在双分子反应中缔合形成二聚体，在20℃时速率常数为4.3×10³ M⁻¹ s⁻¹。第二个限速单分子折叠步骤产生能够进一步缔合的二聚体，在20℃时速率常数为0.5×10⁻³ s⁻¹。四聚体由这些二聚体通过快速反应形成。通过确定β-半乳糖苷酶片段α-互补的类似机制，可以证实β-半乳糖苷酶遵循连续的双单分子折叠和缔合机制。
