Niemirowicz Gabriela, Parussini Fabiola, Agüero Fernán, Cazzulo Juan J
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de General San Martín-CONICET, Av. General Paz 5445, 1650 San Martín, Buenos Aires, Argentina.
Biochem J. 2007 Jan 15;401(2):399-410. doi: 10.1042/BJ20060973.
MCPs (metallocarboxypeptidases) of the M32 family of peptidases have been identified in a number of prokaryotic organisms, and only a few of them have been characterized biochemically. Members of this family are absent from eukaryotic genomes, with the remarkable exception of those of trypanosomatids. The genome of the CL Brener clone of Trypanosoma cruzi, the causative agent of Chagas' disease, encodes two such MCPs, with 64% identity between them: TcMCP-1 and TcMCP-2. Both genes, which are present in a single copy per haploid genome, were expressed in Escherichia coli as catalytically active polyHis-tagged recombinant enzymes. Despite their identity, the purified TcMCPs displayed marked biochemical differences. TcMCP-1 acted optimally at pH 6.2 on FA {N-(3-[2-furyl]acryloyl)}-Ala-Lys with a K(m) of 166 muM. Activity against benzyloxycarbonyl-Ala-Xaa substrates revealed a P1' preference for basic C-terminal residues. In contrast, TcMCP-2 preferred aromatic and aliphatic residues at this position. The K(m) value for FA-Phe-Phe at pH 7.6 was 24 muM. Therefore the specificities of both MCPs are complementary. Western blot analysis revealed a different pattern of expression for both enzymes: whereas TcMCP-1 is present in all life cycle stages of T. cruzi, TcMCP-2 is mainly expressed in the stages that occur in the invertebrate host. Indirect immunofluorescence experiments suggest that both proteins are localized in the parasite cytosol. Members of this family have been identified in other trypanosomatids, which so far are the only group of eukaryotes encoding M32 MCPs. This fact makes these enzymes an attractive potential target for drug development against these organisms.
肽酶M32家族的金属羧肽酶（MCPs）已在多种原核生物中被鉴定出来，其中只有少数经过了生化特性分析。真核生物基因组中没有这个家族的成员，但锥虫属生物是个显著的例外。恰加斯病的病原体克氏锥虫CL Brener克隆株的基因组编码两种这样的MCPs，它们之间有64%的同一性：TcMCP-1和TcMCP-2。这两个基因在每个单倍体基因组中都是单拷贝存在，在大肠杆菌中表达为具有催化活性的多组氨酸标签重组酶。尽管它们具有同一性，但纯化后的TcMCPs显示出明显的生化差异。TcMCP-1在pH 6.2时对FA{N-(3-[2-呋喃基]丙烯酰)}-丙氨酰-赖氨酸的催化活性最佳，米氏常数（K(m)）为166μM。对苄氧羰基-丙氨酰-Xaa底物的活性表明，它对碱性C末端残基有P1'偏好。相比之下，TcMCP-2在这个位置更喜欢芳香族和脂肪族残基。在pH 7.6时，FA-苯丙氨酰-苯丙氨酸的K(m)值为24μM。因此，两种MCPs的特异性是互补的。蛋白质印迹分析显示这两种酶的表达模式不同：TcMCP-1存在于克氏锥虫的所有生命周期阶段，而TcMCP-2主要在无脊椎动物宿主中出现的阶段表达。间接免疫荧光实验表明这两种蛋白质都定位于寄生虫的细胞质中。这个家族的成员已在其他锥虫属生物中被鉴定出来，到目前为止，它们是唯一编码M32 MCPs的真核生物群体。这一事实使得这些酶成为针对这些生物进行药物开发的一个有吸引力的潜在靶点。
