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Originally published In Press as doi:10.1074/jbc.M008702200 on October 19, 2000

J. Biol. Chem., Vol. 276, Issue 2, 1152-1155, January 12, 2001
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Analysis of the Subsite Specificity of Rat Insulysin Using Fluorogenic Peptide Substrates*

Eun-Suk SongDagger , Atish MukherjeeDagger , Maria Aparecida Juliano§, Jan St. Pyrek, Jack P. Goodman Jr., Luiz Juliano§, and Louis B. HershDagger ||

From the Dagger  Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084,  Mass Spectrometry Facility, University of Kentucky, Lexington, Kentucky 40506-0286, and the § Department of Biophysics, Escola Paulista de Medicina, Sao Paulo 04034, Brazil

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.

* This work was supported in part by National Institute on Drug Abuse Grants DA02243 and DA 07062.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Biochemistry, College of Medicine, Chandler Medical Center, University of Kentucky, 800 Rose St., Lexington, KY 40536-0084. Tel.: 859-323-5549; Fax: 859-323-1727; E-mail: lhersh@pop.uky.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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