Venkatesha B, Udgaonkar J B, Rao N A, Savithri H S
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Biochim Biophys Acta. 1998 Apr 23;1384(1):141-52. doi: 10.1016/s0167-4838(98)00013-2.
Equilibrium unfolding studies of the tetrameric serine hydroxymethyltransferase from sheep liver (SHMT, E.C.2.1.2.1) revealed that the holoenzyme, apoenzyme and the sodium borohydride-reduced holoenzyme had random coil structures in 8 M urea. In the presence of a non-ionic detergent, Brij-35, and polyethylene glycol, the 8 M urea unfolded protein could be completely (> 95%) refolded by a 20-fold dilution. The refolded enzyme was completely active and kinetically similar to the native enzyme. The midpoint of inactivation of the enzyme occurred at a urea concentration that was much below the urea concentration required to bring about a substantial loss of secondary structure. This observation suggested the occurrence of a 'predenaturation transition' in the unfolding pathway. The equilibrium urea-induced denaturation curve of holoSHMT showed two transitions. The midpoint of the first transition was 1.2 M, which was comparable to that required for 50% decrease in enzyme activity. Further, 50% release of the pyridoxal-5'-phosphate (PLP) from the active site, as monitored by decrease in absorbance at 425 nm, also occurred at about 1.2 M urea. Size exclusion chromatography showed that the tetrameric SHMT unfolds via the intermediate formation of dimers. This dissociation occurred at a much lower urea concentration (0.15 M) in the unfolding of the apoenzyme, and at a higher urea concentration (1.2 M) in the unfolding of holoenzyme, thereby demonstrating the involvement of PLP in stabilizing the quaternary structure of the enzyme. Size exclusion chromatography of the refolding intermediates demonstrated that the cofactor shifts the equilibrium towards the formation of the active tetramer. The reduced holoenzyme could also be refolded to its native structure, as observed by fluorescence and CD measurements, indicating that the presence of covalently linked PLP does not affect refolding. The results demonstrate clearly that the dimer is an intermediate in the urea-induced equilibrium unfolding/refolding of sheep liver SHMT; and PLP, in addition to its role in catalysis, is required for the stabilization of the tetrameric structure of the enzyme.
对来自绵羊肝脏的四聚体丝氨酸羟甲基转移酶（SHMT，E.C.2.1.2.1）进行的平衡去折叠研究表明，全酶、脱辅酶和硼氢化钠还原的全酶在8 M尿素中具有无规卷曲结构。在非离子去污剂Brij - 35和聚乙二醇存在的情况下，8 M尿素展开的蛋白质通过20倍稀释可完全（> 95%）重新折叠。重新折叠的酶完全有活性，并且在动力学上与天然酶相似。酶失活的中点出现在尿素浓度远低于导致二级结构大量丧失所需的尿素浓度时。这一观察结果表明在去折叠途径中发生了“预变性转变”。全酶SHMT的平衡尿素诱导变性曲线显示出两个转变。第一个转变的中点为1.2 M，这与酶活性降低50%所需的浓度相当。此外，通过425 nm处吸光度的降低监测到，活性位点处50%的磷酸吡哆醛（PLP）释放也发生在约1.2 M尿素时。尺寸排阻色谱表明，四聚体SHMT通过二聚体中间体的形成而展开。这种解离在脱辅酶展开时发生在低得多的尿素浓度（0.15 M）下，而在全酶展开时发生在较高的尿素浓度（1.2 M）下，从而证明了PLP在稳定酶的四级结构中起作用。对重新折叠中间体的尺寸排阻色谱表明，辅因子使平衡向活性四聚体的形成方向移动。通过荧光和圆二色性测量观察到，还原的全酶也可以重新折叠成其天然结构，表明共价连接的PLP的存在不影响重新折叠。结果清楚地表明，二聚体是尿素诱导的绵羊肝脏SHMT平衡去折叠/重新折叠过程中的中间体；并且PLP除了其催化作用外，对于稳定酶的四聚体结构也是必需的。
