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

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）的活性。MPP处理所需的底物中的元素与先前使用以大鼠苹果酸脱氢酶（MDH）延伸肽为模型的合成肽的动力学研究中获得的元素基本相同。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。远端和近端氨基酸之间的部分被证明是灵活的有效处理。我发现MPP通过两组氢键和一个离子键来识别近端精氨酸。从MPP的突变中，β亚基被确定为催化亚基。我成功地表达和纯化了这两个亚基的重组酶。亚基的光亲和标记表明，底物以亚基的不同部分与不同亚基接触的方式与这两种亚基结合。

项目成果

Ogishima,T.: "Analysis of Elements in the Substrate Reqired for Processing by Mitochondrial Processing Peptidase." J.Biol.Chem.270. 30322-30326 (1995)

Ogishima,T.：“线粒体加工肽酶加工所需底物中的元素分析。”

荻島正: "Analysis of Elements in the Substrate Required for Processing by Mitochondrial Processing Peptidase" Journal of Biological Chemistry. 270. 30332-30326 (1995)

Tadashi Ogishima：“线粒体加工肽酶加工所需底物中的元素分析”《生物化学杂志》270. 30332-30326 (1995)。

Kitada, S., Shimokata, K., Niidome, T., Ogishima, T., & Ito, A.: "A Putative Metal-binding Site in the beta Subunit of Rat Mtochondrial Processing Peptidase is Essential for Its Catalytic Activity" J.Biochem.117. 1148-1150 (1995)

北田，S.，下方，K.，新留，T.，扇岛，T.，

北田栄: "A Putative Metal-Binding Site in the β-Subunit of Rat Mitochondrial Processing Peptidase is Essential for Its Catalytic Activity" Journal of Biochemistry. 117. 1148-1150 (1995)

Sakae Kitada：“大鼠线粒体加工肽酶 β 亚基中的假定金属结合位点对其催化活性至关重要”，生物化学杂志 117。1148-1150 (1995)。

Song,M-C.: "Role of the Basic Amino Acids in the Cleavage of Synthetic Peptide Substrates by Mitochondrial Processing Peptidase" J.Biochem.120. 1163-1166 (1996)

Song,M-C.：“碱性氨基酸在线粒体加工肽酶裂解合成肽底物中的作用”J.Biochem.120。