Whole Statue of Recognition by Enzyme Toward Substarates with Vague Signals

(1)使用荧光共振能量转移测量（FRET）获得结构信息。合成了一系列在远端和近端精氨酸之间具有不同插入长度的肽底物，然后与荧光受体和供体共价连接。当底物与酶结合时，探针之间的距离基本相同。当在底物和酶之间进行FRET时，也获得了这样的共同长度。(2)在许多前体蛋白中，一些蛋白质缺少近端精氨酸残基。这些前体仍然被线粒体加工肽酶（MPP）切割。当末端残基改为精氨酸时，加工效率略有提高。这表明S2-P2位置之间仍然存在一些相互作用。(3)MPP显示出切割融合蛋白的内部肽键。虽然融合蛋白已经建立了独立的结构，但MPP可以在一个结构松散的结构域中攻击肽池。(4)使用FRET测量研究MPP与切割位点以外的区域周围的底物之间的相互作用。结果表明，在切割位点下游约10个氨基酸的部分可以从MPP口袋中出现。

(1) Structural information was obtained using fluorescence resonance energy transfer measurement (FRET). A series of the peptide substrate with different intervening lengths between the distal and proximal argines were synthesized and then covalently attached with the fluorescence acceptor and donnaor. When the substrates were bound to the enzyme, essentially the same distances between the probes were obtained. Such common length was also obtained when when FRET was conducted between the substrates and enzyme.(2) Among many precursor proteins some proteins lack the proximal arginine residue. Such precursors were still cleaved by mitochondrial processing peptidase (MPP). When the distal residue was changed to arginine, the processing efficiency was slightly elevated. This indicates that some interactions are still present between the S2-P2 positions.(3) MPP was shown to cleave the internal peptide bond of a fusion protein. Although the fusion protein had established the individual structure, MPP could attacked the peptide pond in a loosely structured domain.(4) The interaction between MPP and the substrates around regions other than the cleavage site was investigated using FRET measurement. The results indicated that a portion downstream from the cleavage site about 10 amino acids could emerge from the MPP pocket.