Mizobata T, Akiyama Y, Ito K, Yumoto N, Kawata Y
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
J Biol Chem. 1992 Sep 5;267(25):17773-9.
The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE. Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer. The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not. Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation. The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL. However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well. Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well. We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules. The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations.
伴侣蛋白GroE促进了四聚体酶色氨酸酶的重折叠。在GroE 21聚体比色氨酸酶单体过量15倍的情况下，色氨酸酶分子的重折叠产率达到最大值（约80%）。重折叠产率的提高需要GroEL亚基，而GroES亚基则不需要。重折叠反应的光散射实验表明，GroE与色氨酸酶折叠中间体结合并抑制其聚集。色氨酸酶从GroEL上有效解离需要ATP的存在。然而，我们的实验表明，色氨酸酶不仅在ATP存在下，而且在不可水解的ATP类似物如ATPγS（腺苷5'-O-（3-硫代三磷酸））和AMP-PNP（腺苷-5'-基亚氨基二磷酸）存在下也能很容易地从GroEL上解离。令人惊讶的是，在ADP存在下色氨酸酶从GroEL上的释放也得到促进。我们得出结论，ATP和ADP等核苷酸的结合改变了GroEL的构象，促进了色氨酸酶分子的解离。如各种折叠蛋白从这两种构象中的选择性解离所示，在ADP存在下形成的构象与在ATP存在下形成的构象不同。
