Sequential domain refolding of pig muscle 3-phosphoglycerate kinase: kinetic analysis of reactivation

Andrea N. Szilégyi , Méria Vas
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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.

猪肌3-磷酸甘油酸激酶序列结构域重折叠:再激活动力学分析
背景:3-磷酸甘油酸激酶的缓慢再折叠被认为主要是由其结构域结构引起的:c端结构域的折叠和/或结构域配对被认为是限速步骤。在猪肌酶分离的c端蛋白水解片段(比c结构域大约22 kDa乘5 kDa)的重折叠过程中,观察到一个缓慢的异构化。本文主要研究了这一步骤在酶类重组中的作用。结果:3-磷酸甘油酸激酶或其互补的蛋白水解片段(残基1-155和156-416)在重折叠期间的再激活时间过程表现出明显的滞后期,表明形成了非活性折叠中间体。整个过程可以用两个连续的一阶步骤(速率常数分别为0.012±0.0035和0.007±0.0020 s−1)来描述,导致酶活性的高回收率(60-85%)。c片段的预先还原恢复了MgATP与c结构域的结合,并取消了较快的步骤,允许单独观察较慢的步骤。据此,通过色氨酸荧光变化监测到的c结构域的重折叠发生的速率与较快步骤相似。结论:除了之前观察到的c结构域中的缓慢重折叠步骤(0.012 s−1)外,还检测到另一个可能在n结构域中的缓慢重折叠步骤(0.007 s−1)。c结构域的独立折叠被证明,然而,从比较动力学分析,n结构域的独立折叠看起来不太可能。我们的数据更符合一个顺序的机制,而不是随机的机制,并表明首先发生c结构域的折叠,导致一个非活性中间体,然后是n结构域的折叠。
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