Lowry C L, McGeehan G, LeVine H
Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
Proteins. 1992 Jan;12(1):42-8. doi: 10.1002/prot.340120106.
A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.
从大肠杆菌包涵体中纯化出一种重组19 kDa人成纤维细胞胶原酶催化片段，该片段以天然存在的蛋白水解产物为模型。在锌和钙存在下复性后，该片段对小的合成底物表现出与全长胶原酶相同的一级序列特异性催化活性。与亲本酶不同，它能快速裂解酪蛋白和明胶，但不能裂解天然I型胶原。利用三个色氨酸残基的固有荧光来监测酶的构象状态，变性时其发射光谱发生24 nm红移，同时伴随着荧光猝灭和催化活性丧失。低浓度变性剂可使该片段展开，而全长酶则呈现出平缓的延伸变性曲线。钙能显著稳定19 kDa片段，锌的作用稍弱，而二者共同作用时具有协同稳定作用。在二价阳离子中，钙是最有效的稳定剂，EC50约为60 μM，底物水解所需的钙量与之相似。催化活性比色氨酸荧光对变性更敏感。通过圆二色性评估的多肽主链二级结构最不敏感。这些观察结果表明，19 kDa胶原酶片段的折叠是一个由钙稳定的多步过程。
