Parker M J, Spencer J, Jackson G S, Burston S G, Hosszu L L, Craven C J, Waltho J P, Clarke A R
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochemistry. 1996 Dec 10;35(49):15740-52. doi: 10.1021/bi961330s.
Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monomeric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain alpha-helix (residues 170-185). The molecule folds to the native state in three stages. In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol-1 and the C-domain by 3.3 kcal mol-1 over their respective unfolded conformations. The N-domain then converts to a folded state at a rate of 1.2 s-1 (delta GI-F = 3.8 kcal mol-1), followed by the C-domain at 0.032 s-1 (delta GI-F = 12.1 kcal mol-1). It is this last step that limits the rate of acquisition of enzyme activity. In the dynamics of unfolding in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s-1, some 10(7) times faster than the C-domain. Consequently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a native-like C-domain. In a conventional sense, therefore, the folding/unfolding kinetics of bsPGK can be described as random order. Consistent with these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394. A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relatively weak, contributing approximately 1.4 kcal mol-1 to the stability of the active enzyme. The only interactions which contribute to the stability of rapidly formed intermediates or to transition states along the productive folding pathways are those within domain cores. Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but do not constitute a separate step in the folding mechanism. Intriguingly, the most pronounced effect of interdomain contacts on the kinetics of folding is inhibitory; the presence of the C-domain appearing to reduce the effective rate of acquisition of native structure within the N-domain.
嗜热栖热芽孢杆菌磷酸甘油酸激酶（bsPGK）是一种由394个残基组成的单体酶，包含两个球状结构域（N和C），通过一个结构域间的α螺旋（残基170 - 185）共价连接。该分子在三个阶段折叠成天然状态。在第一阶段，每个结构域迅速且独立地折叠形成一个中间体，其中N结构域相对于其各自的未折叠构象稳定化程度为5.1千卡/摩尔，C结构域为3.3千卡/摩尔。然后，N结构域以1.2秒^-1的速率转变为折叠状态（ΔG_I - F = 3.8千卡/摩尔），接着C结构域以0.032秒^-1的速率转变（ΔG_I - F = 12.1千卡/摩尔）。正是这最后一步限制了酶活性获得的速率。在水中展开的动力学过程中，N结构域以8×10^(-4)秒^-1的速率转变为中间状态，比C结构域快约10^7倍。因此，折叠反应中最主要的中间体在N结构域具有类似天然的结构，而在展开方向上的中间体在C结构域具有类似天然的结构。所以，从传统意义上讲，bsPGK的折叠/展开动力学可以描述为随机顺序。与这些观察结果一致，在结构域间螺旋处切割该分子会产生两个独立稳定的单元，分别包含残基1 - 175和180 - 394。将它们的折叠行为与整个分子的折叠行为进行详细比较表明，真正的结构域间接触相对较弱，对活性酶的稳定性贡献约为1.4千卡/摩尔。对沿着有效折叠途径快速形成的中间体或过渡态稳定性有贡献的唯一相互作用是结构域核心内的相互作用。结构域之间或与结构域间螺旋形成的接触仅在折叠的基态中形成，但不构成折叠机制中的一个独立步骤。有趣的是，结构域间接触对折叠动力学最显著的影响是抑制性的；C结构域的存在似乎降低了N结构域内天然结构有效获得的速率。
