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Nichtl, Alfons ; Buchner, Johannes ; Jaenicke, Rainer ; Rudolph, Rainer ; Scheibel, Thomas:
Folding and association of β-galactosidase.
In: Journal of Molecular Biology.
Bd. 282
(1998)
Heft 5
.
- S. 1083-1091.
ISSN 0022-2836
DOI: https://doi.org/10.1006/jmbi.1998.2075
Abstract
β-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 β-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 4.3×103 M−1 s−1at 20°C. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5×10−3 s−1at 20°C. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for α-complementation of β-galactosidase fragments it could be confirmed that β-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.