Analysis of the subsite specificity of rat insulysin using fluorogenic pepticle substrates

Eun Suk Song, Atish Mukherjee, Maria Aparecida Juliano, Jan St Pyrek, Jack P. Goodman, Luiz Juliano, Louis B. Hersh I

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35 Scopus citations


Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a Km of 13 μM and a Vmax of 2.6 μmol min-1 mg-1. Derivatives of this peptide in which the P2 leucine or the P2′ valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P2 residue produced a 4-fold range in Km and a 7-fold range in kcat. The nature of the P2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P2′ residues produce less of a change in both Km and kcat and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.

Original languageEnglish
Pages (from-to)1152-1155
Number of pages4
JournalJournal of Biological Chemistry
Issue number2
StatePublished - Jan 12 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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