Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation.
Treatment of an N-terminal-containing His6-tagged insulysin (His6-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of approximately 95 and approximately 76 kDa and finally a relatively stable approximately 56 kDa fragment. The approximately 76 and approximately 56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE approximately 76 and approximately 56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His6-IDE. The approximately 76 and approximately 56 kDa IDE proteolytic fragments were active toward the physiological peptides beta-endorphin, insulin, and amyloid beta peptide 1-40. Although activity was reduced by a factor of approximately 103-104 with these substrates, the relative activity and the cleavage sites were unchanged. Both the approximately 76 and approximately 56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.
Song
Eun Suk
ES
Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, 741 South Limestone, B283 Biomedical Biological Sciences Research Building, Lexington, Kentucky 40536-0509, USA.
Cady
Clint
C
Fried
Michael G
MG
Hersh
Louis B
LB
D000494:Allosteric Regulation; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D019286:Endopeptidase K / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry*; D007339:Insulysin / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D001615:beta-Endorphin / Q000737:chemistry*
D016229:Amyloid beta-Peptides; D007328:Insulin; D010455:Peptides; D011994:Recombinant Proteins; D001615:beta-Endorphin; D019286:Endopeptidase K; D007339:Insulysin