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

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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.