Nguyen Duy Duc, Pandian Ramesh, Kim Doyoun, Ha Sung Chul, Yoon Hye-Jin, Kim Kap Sun, Yun Kyung Hee, Kim Jin-Hahn, Kim Kyeong Kyu
Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
Pohang Acceleratory Laboratory, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea.
Biochem Biophys Res Commun. 2014 Apr 25;447(1):101-7. doi: 10.1016/j.bbrc.2014.03.109. Epub 2014 Apr 1.
The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co(2+)- and Zn(2+)-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co(2+) for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.
MH家族中的肽酶被认为是在活性位点含有两个锌离子的共催化锌肽酶，但其金属偏好性尚未得到严格研究。在本研究中，通过结构和生化分析提供了金属偏好性的分子基础。对属于MH家族中肽酶家族M18的铜绿假单胞菌天冬氨酰氨基肽酶（PaAP）的动力学研究表明，其肽酶活性依赖于Co(2+)而非Zn(2+)：在无金属、Co(2+)和Zn(2+)条件下，PaAP的kcat（s(-1)）值分别为0.006、5.10和0.43。一致地，向预先用Zn(2+)饱和的PaAP中添加低浓度的Co(2+)可大大增强酶活性，这表明Co(2+)可能是PaAP生理相关的共催化金属离子。PaAP与Co(2+)或Zn(2+)复合物的晶体结构通常显示活性位点中有两个金属离子与三个保守残基和一个四方几何结构中的碳酸氢根离子配位。然而，除了活性位点中的通用碱Glu-265的相对取向外，结合Co(2+)和Zn(2+)的结构未显示与金属种类的差异效应相关的明显变化。突变体PaAP的表征表明，第一个金属结合位点主要负责金属偏好性。与PaAP类似，属于MH家族中氨基肽酶家族M42的肺炎链球菌谷氨酰氨基肽酶（SpGP）也显示最大活性需要Co(2+)。这些结果表明，MH家族肽酶可能是共催化钴肽酶而非锌依赖性肽酶。
