Document Type
Peer-Reviewed Article
Publication Date
8-14-2018
Abstract
Insulin-degrading enzyme (IDE) is a 110 kDa chambered zinc metalloendopeptidase that degrades insulin, amyloid beta, and other intermediate-sized aggregation prone peptides that adopt β-structures. Structural studies of IDE in complex with multiple physiological substrates have suggested a role for hydrophobic and aromatic residues of the IDE active site in substrate binding and catalysis. Here, we examine functional requirements for conserved hydrophobic and aromatic IDE active site residues that are positioned within 4.5 Angstroms of IDE bound insulin B chain and amyloid beta peptides in the reported crystal structures for the respective enzyme-substrate complexes. Charge, size, hydrophobicity, aromaticity, and other functional group requirements for substrate binding IDE active site residues were examined through mutational analysis of the recombinant human enzyme and enzyme kinetic studies conducted using native and fluorogenic derivatives of human insulin and amyloid beta peptides. A functional requirement for IDE active site residues F115, A140, F141, Y150, W199, F202, F820, and Y831 was established, and specific contributions of residue charge, size and hydrophobicity in substrate binding, specificity, and proteolysis were demonstrated. IDE mutant alleles that exhibited enhanced or diminished proteolytic activity towards insulin or amyloid beta peptides and derivative substrates were identified.
DOI
10.1021/acs.biochem.8b00474
PubMed ID
30004674
Recommended Citation
Stefanidis, L., Fusco, N. D., Cooper, S. E., Smith-Carpenter, J., & Alper, B. J. (2018). Molecular determinants of substrate specificity in human insulin-degrading enzyme. Biochemistry, 57(32), 4903-4914. doi: 10.1021/acs.biochem.8b00474
Included in
Amino Acids, Peptides, and Proteins Commons, Chemistry Commons, Enzymes and Coenzymes Commons
Comments
Version posted is the "Just Accepted" manuscript.
Lazaros Stefanidis is a graduate student in the Chemistry Department at Sacred Heart University. Nicholas D. Fusco is an undergraduate student in the Chemistry Department at Sacred Heart University.