Keynan S, Hooper N M, Turner A J
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem J. 1997 Aug 15;326 ( Pt 1)(Pt 1):47-51. doi: 10.1042/bj3260047.
Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.
膜二肽酶（EC 3.4.13.19）是一种质膜锌肽酶，参与谷胱甘肽及其共轭物（如白三烯D4）的肾脏代谢。该酶缺乏其他锌依赖性水解酶的经典特征，且与任何其他哺乳动物蛋白均无同源性。我们利用定点诱变技术探究了猪膜二肽酶中五个在哺乳动物物种间保守的组氨酸残基的作用。当在COS-1细胞中表达时，突变体H49K和H128L对底物甘氨酰-D-苯丙氨酸的比活性和Km值与野生型酶相当。然而，突变体H20L、H152L和H198K无活性，但通过免疫印迹和免疫荧光评估，它们在细胞表面的表达水平与野生型相当。比较了这三个突变体与竞争性抑制剂西司他丁结合的能力，西司他丁与天然的和经乙二胺四乙酸处理的猪肾膜二肽酶结合效率相同。表达的野生型酶以及突变体H20L和H198K能有效地与西司他丁-琼脂糖结合，但H152L未能结合。因此，His-152似乎参与底物或抑制剂的结合，而His-20和His-198似乎参与催化作用。膜二肽酶与最近从醋酸钙不动杆菌克隆的一种二肽酶有一些相似之处。特别是，膜二肽酶的His-20和His-198在该细菌酶中保守，此前已证明具有催化活性所需的Glu-125和His-219也保守。
