Szilágyi A N, Vas M
Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Budapest.
Fold Des. 1998;3(6):565-75. doi: 10.1016/s1359-0278(98)00071-6.
Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated.
The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1-155 and 156-416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60-85%) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012+/-0.0035 and 0.007+/-0.0020 s(-1)). A prior renaturation of the C-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step.
In addition to the previously observed slow refolding step (0.012 s(-1)) within the C-domain, the occurrence of another slow step (0.007 s(-1)), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.
