Analysis for the Substrate Strucure and Structure Responsible for Substrate-recognition by Mitochondrial Processing Peptidase

底物结构和线粒体加工肽酶识别底物的结构分析

• 批准号: 07680692
• 负责人: OGISHIMA Tadashi
• 金额: $ 1.22万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680692/
• 关键词: Processing Peptidase; Protease; Substrate-recognition; Photoaffinity Labelling; プロセシングプロテアーゼ; ペプチド基質; ミトコンドリアタンパク質前駆体; 延長ペプチド; 分子認識; フォトアフィニティーラベル; Processing Peptidase; Protease; Substrate-recognition; Photoaffinity Labelling; プロセシングプロテアーゼ; ペプチド基質; ミトコンドリアタンパク質前駆体; 延長ペプチド; 分子認識; フォトアフィニティーラベル

I developed fluorogenic peptides, which were applicable for a rapid and real-time measurement of the acitivity of mitochondrial processing peptidase (MPP). Elements in the substrate required for processing by MPP were essentially the same as obtained from the previous kinetical study using synthetic peptides modeled on the extension peptides of rat malate dehydrogenase (MDH). Primary recognition elements were the distal basic amino acid and proximal arginine residues, and the amino acid at P1' position. The portion between the distal and proximal amino acids proved to be flexible for the efficient processing. I found that MPP recognizes the proximal arginine via two sets of hydrogen-bonding and one ionic bonding.From a mutation of MPP,beta-subunit was determined to be the catalytic subunit. I succeeded in expressing and purifying recombinant enzyme of both subuits. Photoaffinity labelling of the subuints demonstrated that the substrate binds to both subuints in the manner that different portions of the substate were in contact with different subunits.

我开发了荧光肽，可用于快速，实时测量线粒体加工肽酶（MPP）的阳性。 MPP加工所需的底物中的元素基本与从先前的Kination研究中使用的合成肽在大鼠苹果酸脱氢酶（MDH）的延伸肽上获得的元素相同。主要识别元素是远端碱性氨基酸和近端精氨酸残基，以及P1'位置的氨基酸。事实证明，远端和近端氨基酸之间的部分对于有效的加工是灵活的。我发现MPP通过两组氢键和一组离子键识别近端精氨酸。从MPP突变，β-亚基被确定为催化亚基。我成功地表达并净化了这两个亚装的重组酶。子量的光性标记表明，底物以两种取代的方式与不同的亚基接触的方式与两个子图结合。