Shinde U, Fu X, Inouye M
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
J Biol Chem. 1999 May 28;274(22):15615-21. doi: 10.1074/jbc.274.22.15615.
Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer's disease. The molecular basis of such diversity is unknown. Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed "protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by Ile-48 --> Thr IMC (IMCI-48T) acquires an "altered" enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates. IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin. Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC.
在诸如羊瘙痒症和阿尔茨海默病等疾病中，可观察到独特氨基酸序列内的构象多样性。这种多样性的分子基础尚不清楚。类似的现象也发生在枯草杆菌蛋白酶中，它是一种与真核生物激素原转化酶同源的丝氨酸蛋白酶。枯草杆菌蛋白酶原肽作为一种分子内伴侣（IMC），在折叠过程中赋予空间信息，但酶活性并不需要它。IMC内的点突变会改变折叠，导致结构构象异构体在一个被称为“蛋白质记忆”的过程中与其同源IMC发生特异性相互作用。在这里，我们展示了一种介导枯草杆菌蛋白酶构象多样性的机制。在成熟过程中，当IMC被枯草杆菌蛋白酶的活性位点自切割并随后降解时，该位点切割前后的酶学性质有显著差异。尽管由Ile-48→Thr IMC（IMCI-48T）折叠的枯草杆菌蛋白酶获得了一种与野生型枯草杆菌蛋白酶（SubWT）显著不同的“改变的”酶活性构象（SubI-48T），但两种前体以相似的速率进行自切割。IMC切割引发构象变化，在此期间，IMC从枯草杆菌蛋白酶切割后继续其伴侣功能。导致构象多样性的结构印记起源于这个重组阶段，是由IMC自切割催化的后期折叠事件。
