Davy A, Svendsen I, Sørensen S O, Blom Sørensen M, Rouster J, Meldal M, Simpson D J, Cameron-Mills V
Carlsberg Research Laboratory, Department of Chemistry and Department of Physiology, Carlsberg Laboratory, Gammel Carlsbergvej 10, DK-2500 Valby, Denmark.
Plant Physiol. 1998 May;117(1):255-61. doi: 10.1104/pp.117.1.255.
The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1'-P2'-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1', showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1' greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower Km values.
从绿大麦（Hordeum vulgare L.）麦芽中纯化出半胱氨酸内肽酶（EP）-A和EP-B，并通过N端氨基酸测序确认了它们的身份。通过对切割产物进行N端氨基酸测序确定了重组C型大麦醇溶蛋白中EP-B的切割位点，并用于设计内部淬灭的荧光肽底物。通式为2-氨基苯甲酰基-P2-P1-P1'-P2'-酪氨酸（NO2）-天冬氨酸的四肽底物（其中切割发生在P1和P1'之间）表明，半胱氨酸内肽酶在P2处优先选择苯丙氨酸、亮氨酸或缬氨酸。在P1处，精氨酸比谷氨酰胺更受青睐，而在P2、P1或P1'处的脯氨酸会大大降低底物的动力学特异性。C型大麦醇溶蛋白的酶切主要由切割位点的一级序列决定，因为基于C型大麦醇溶蛋白序列延长底物并不会使它们成为更合适的底物。用丝氨酸或脯氨酸取代亮氨酸对C型大麦醇溶蛋白进行定点诱变，破坏了主要切割位点。EP-A和EP-B的活性均高于木瓜蛋白酶，这主要是因为它们的Km值低得多。
