Mechanism of Processing of Mitochondrial Protein Precursor

线粒体蛋白前体的加工机制

• 批准号: 08458200
• 负责人: ITO Akio
• 金额: $ 5.5万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458200/
• 关键词: processing peptidase; mitochondria; processing; precursor protein; substrate recognition; peptide substrate; fluorescent probe; プロセシングペプチターゼ; 基質認識; プロセシングペピチダーゼ

Mitochondrial processing peptodase (MPP) specifically recognaizes large variety of mitochondrial precursorproteins and cleaves off amino-terminal extension peptides. The enzymeforms a heterodimerconsisting of structurally related alpha-and beta-subunits. Structure common to all the extension peptides that is requiredfor specific recognition by MPP and role of each subunit in catalytic reaction and substrate recognition were studied.1. Two sets of basic amino acids in the peptide, the proximal arginine residue at position-2 and the distal basic residues at positions about-10 relative to the cleavagesite, and hydrophobicamino acids at position 1 are necessary for effective hydrolysis. Residues position 2 and 3 also contribute significantly to the efficiency of processing.2.To investigate the responsibility of MPP subunits for substrate recognition, we used the fluorescent-labeled peptide substrates with a coumarin derivative and found that MPP bound the substrate peptides with high affin … More ity (Kd ; 0.13muM) only in the dimericcomplex and each subunit had about a 30-foldless affinity than the somplex, indicating that the two subunits of MPP cooperatively form a substrate-binding pocket.3.Mutation experiments demonstrate that three glutamate residues, positions at 79,129, and 136, around the metal binding sitein beta-MPP,in addition to a putative active site glutamate at position 59, are essential for the function. Glu-79,129, and 136 appears to be involved in substrate binding, catalysis, and metal binding, respectively.4.Mutation of acidic amino acidresidues in the carboxy-terminal portion of the alpha-MPP causes a decreasein cleavage efficiency of precursors with a longer extension peptide.Taken together, we proposed a model for substrate recogmition of MPP,in which the two subunits of MPP cooperatively form the substrate binding pocket and recognize different structural elements in the extension peptide. The multiple-site recognition mechanism may make it possible to render strict spesificity and high affinity to MPP for precursors with structures little in common. Less

线粒体加工肽酶(MPP)能特异性识别多种线粒体前体蛋白，并能切割出氨基末端延伸肽。该酶形成由结构上相关的α-和β-亚基组成的异二聚体。研究了MPP特异性识别所需的所有延伸肽的共同结构，以及各亚基在催化反应和底物识别中的作用。多肽中的两组碱性氨基酸，即-2位的近端精氨酸残基和-10位的远端碱性残基，以及1位的疏水氨基酸是有效水解所必需的。2.为了研究mpp亚单位对底物识别的作用，我们使用带有香豆素衍生物的荧光标记的多肽底物，发现mpp与高亲和…的底物结合3.突变实验表明，在β-MPP中，金属结合部位周围的79、129和136位的三个谷氨酸残基，以及59位推测的活性部位谷氨酸，对β-MPP的功能是必不可少的。Glu-79、129和136分别参与底物结合、催化和金属结合。4.α-MPP羧基末端的酸性氨基酸残基的突变导致具有较长延伸肽的前体的切割效率降低。多位点识别机制可以使结构几乎没有共同点的前体对MPP表现出严格的快速性和高亲和力。较少

项目成果

伊藤明夫: "ミトコンドリア蛋白質のプロセシング" 蛋白質核酸酵素. 42・14. 2362-2367 (1997)

伊藤昭夫：“线粒体蛋白质的加工”蛋白质核酸酶42・14（1997）。

宋 明哲: "Role of the Basic Amino Acids in the Clevage of Synthetic Peptide Subatrates by Mitochondrial Processing Peptidase" Journal of Biochemistry. 120. 1163-1166 (1996)

Ming-cheol Song：“碱性氨基酸在线粒体加工肽酶裂解合成肽底物中的作用”《生物化学杂志》120。1163-1166 (1996)。

柘野 幸生: "A key amino acid responsible for substrate selectivity of monoamine oxidase A and B" Journal of Bilological Chemistry. 272・22. 14033-14036 (1997)

Yukio Tsuano：“负责单胺氧化酶 A 和 B 的底物选择性的关键氨基酸”《生物化学杂志》272・22（1997 年）。

下方 国稔: "Substrate recoginition by mitochondrial processing peptidase toward the malate dehydrogenase precursor" Journal of Biochemistry. 122・5. 1019-1023 (1997)

Kuninoshi Shimogata：“线粒体加工肽酶对苹果酸脱氢酶前体的底物识别”《生物化学杂志》122・5（1997）。

伊藤明夫: "Recognition of the precursor proteins by the mitochondrial processing peptidase" Proteolysis in Cell Functions. 332-339 (1997)

Akio Ito：“线粒体加工肽酶对前体蛋白的识别”《细胞功能中的蛋白水解》332-339 (1997)。