{"title":"Sequential domain refolding of pig muscle 3-phosphoglycerate kinase: kinetic analysis of reactivation","authors":"Andrea N. Szilégyi , Méria Vas","doi":"10.1016/S1359-0278(98)00071-6","DOIUrl":null,"url":null,"abstract":"<div><p><strong>Background:</strong> 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.</p><p><strong>Results:</strong> 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<sup>−1</sup>). 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.</p><p><strong>Conclusions:</strong>In addition to the previously observed slow refolding step (0.012 s<sup>−1</sup>) within the C-domain, the occurrence of another slow step (0.007 s<sup>−1</sup>), 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.</p></div>","PeriodicalId":79488,"journal":{"name":"Folding & design","volume":"3 6","pages":"Pages 565-575"},"PeriodicalIF":0.0000,"publicationDate":"1998-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1359-0278(98)00071-6","citationCount":"18","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Folding & design","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1359027898000716","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 18
Abstract
Background: 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.
Results: 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.
Conclusions: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.