Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates.
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 K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) 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 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, 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.
Song
E S
ES
Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
Mukherjee
A
A
Juliano
M A
MA
Pyrek
J S
JS
Goodman
J P
JP
Juliano
L
L
Hersh
L B
LB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004140:Dinitrophenols / Q000493:pharmacokinetics*; D006868:Hydrolysis; D007339:Insulysin / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D004140:Dinitrophenols; D009842:Oligopeptides; D011994:Recombinant Proteins; C422992:glycyl-glycyl-phenylalanyl-leucyl-arginyl-lysyl-valyl-glycyl-glutaminyl-ethylenediamine-2,4-dinitrophenol; D007339:Insulysin