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

Recombinant rat insulysin was shown to cleave the internally quenched fluor ogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K-m of 13 muM and a V-max of 2.6 mu mol min(-1) mg(-1). Derivatives of this peptide in which the P-2 leucine or the P-2' v aline were replaced with other residues were used to probe the subsite spec ificity of the enzyme. Varying the P-2 residue produced a l-fold range in K -m and a 7-fold range in k(cat). The nature of the P-2 residue had a signif icant effect on the site of cleavage. Leucine, isoleucine, valine, and aspa rtate produced cleavage at the R-K bond, Asparagine produced 36% cleavage a t the N-R bond and 64% cleavage at the RK bond, whereas with alanine or ser ine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phe nylalanine, methionine, or histidine representing the varied residue X, cle avages at F-X, X-R, and RK were seen, whereas with tryptophan equal cleavag e occurred at the F-W and W-R bonds. Variable P-2' residues produce less of a change in both K-m and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, whic h in addition to producing cleavage at the RK bond, produce significant cle avage at the GR bond. Alanine and tyrosine were unique in producing cleavag e at the F-L bond. Taken together, these data suggest that insulysin specif icity is directed toward the amino side of hydrophobic and basic residues a nd that the enzyme has an extended substrate binding site.