Vasiljeva Olga, Dolinar Marko, Turk Vito, Turk Boris
Department of Biochemistry and Molecular Biology, Josef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Biochemistry. 2003 Nov 25;42(46):13522-8. doi: 10.1021/bi035355k.
Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.
人组织蛋白酶H在大肠杆菌中以包涵体形式表达。经过复性和自催化激活后，产生了一种缺少小链的重组组织蛋白酶H形式。去除小链完全消除了该酶的氨肽酶活性，并大幅提高了其内切肽酶活性（约40倍）。与组织蛋白酶S类似，Bz - FVR - AMC（k(cat)/K(m)值为1070 mM(-1) s(-1)）被发现是重组组织蛋白酶H的首选底物。然而，在较高底物（Z - FR - AMC、Bz - FVR - AMC）浓度下观察到底物抑制现象。重组组织蛋白酶H对蛋白质底物胰岛素β链也有内切肽酶活性，主要切割位点在Glu13 - Ala14之间。重组人组织蛋白酶H受到鸡半胱氨酸蛋白酶抑制剂、抑半胱氨酸蛋白酶蛋白A和抑半胱氨酸蛋白酶蛋白B的抑制，K(i)值在0.05 - 0.1 nM范围内，这比相同抑制剂对纯化组织蛋白酶H的抑制作用略强。因此，这些结果表明组织蛋白酶H小链对该酶的氨肽酶活性至关重要，但对半胱氨酸蛋白酶抑制剂的抑制作用影响较小。
