Departamento de Bioquímica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Phytochemistry. 2010 Apr;71(5-6):524-30. doi: 10.1016/j.phytochem.2009.12.018. Epub 2010 Jan 29.
The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg(180) located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.
卡拉胶属和番木瓜属植物的晶格包含半胱氨酸蛋白酶同工型，有助于保护这些植物免受伤害。在之前的研究中，我们从卡拉胶属植物中分离出 14 种不同的蛋白质成分，其中两种在哺乳动物细胞中具有有丝分裂活性。在这项研究中，我们比较了具有有丝分裂活性的蛋白酶之一（CMS2MS2）与该组酶活性最高的同工型之一（CMS1MS2）的动力学参数。两种酶对 BAPNA（苯甲酰-精氨酸-PNA）或 PFLPNA（吡咯-苯丙氨酸-亮氨酸-PNA）的 Km 值相似，但 CMS1MS2 对 BAPNA 的 kcat 约高 14 倍，对 PFLPNA 的 kcat 约高 129 倍。虽然两种酶都被 E-64 和碘乙酰胺抑制，但鸡半胱氨酸蛋白酶抑制剂完全抑制 CMS1MS2，但对 CMS2MS2 的活性影响很小。根据这些蛋白质的结构以及其他已解析结构的番木瓜科家族的酶，推测 CMS2MS2 活性部位裂隙中的 Arg（180）负责其对半胱氨酸蛋白酶抑制剂的抗性。
