Tomar Ritu, Dubey Vikash Kumar, Jagannadham M V
Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Biochimie. 2009 Aug;91(8):951-60. doi: 10.1016/j.biochi.2009.04.013. Epub 2009 May 3.
Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to alpha/beta class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (U(P)) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from alpha/beta to beta-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U(P) state occurred in two steps with the formation of beta state at low alcohol concentration followed by stabilization of beta state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.
蛋白酶K（E.C. 3.4.21.64）是一种来自真菌白僵菌的丝氨酸蛋白酶，已被用作模型系统来研究在低pH值下一元醇诱导的构象变化。蛋白酶K属于α/β类蛋白质，在pH 7.0 - 3.0范围内保持结构完整性。在pH 2.5时，酶获得部分未折叠构象（U(P)），活性较低，三级结构部分丧失，一些疏水区域暴露。选择处于pH 2.5应激状态的蛋白酶K，研究烷基醇（甲醇/乙醇/异丙醇）诱导的构象变化。在醇的临界浓度下，蛋白质结构发生构象转换，从α/β转变为β-折叠，使蛋白质进入O态。O态中三级接触和蛋白水解活性的完全丧失强调了α区域在维持酶活性位点中的作用。此外，异丙醇诱导处于U(P)状态的蛋白酶K展开分两步进行，在低醇浓度下形成β态，随后在高醇浓度下β态稳定。盐酸胍和温度诱导处于O态（在50%异丙醇中）的蛋白酶K展开是非协同的，因为转变曲线是双相的。这表明处于O态的蛋白酶K结构中α区域已熔化，β区域已稳定，并且这些差异稳定的区域依次展开。此外，通过添加50%异丙醇可以从完全未折叠的蛋白质获得蛋白酶K的O态。因此，醇诱导的O态不同于天然态或完全未折叠态，表现出类似熔球态的特征。因此，这种状态可能在蛋白酶K的折叠途径中起中间体的作用。
