Targeting proteolysis to non-physiological protein substrates

David R. Corey, Gary S. Coombs, Robert C. Bergstrom, Edwin L. Madison

Research output: Contribution to journalArticlepeer-review


In this study we investigate the introduction of these sequences into the 44-51 loop of staphylococcal nuclease (SNase) to determine whether the sequence specificities that u-PA and t-PA exhibit toward peptide substrates are retained for conformationally constrained sequences within nonphysiologic protein targets. We find that both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences. Km for protein cleavage is reduced up to 330-fold relative to Km for cleavage of analogous sequences within fifteen residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately five fold more strongly than that of trypsin, suggesting that cleavage by t-PA may be more dependent on target site mobility. This dependence is chiefly an effect on Km and may be a general mechanism that helps highly specific proteases achieve their narrow specificity. Cleavage of proteins by t-PA and u-PA is sequence-selective. u-PA is 47 fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, while t-PA is 230 fold more active towards a t-PA-selective sequence. These results suggest that non-Si subsite interactions play an important role in determining both the selectivity and the catalytic efficiency of serine proteases towards protein substrates.

Original languageEnglish (US)
Pages (from-to)51
Number of pages1
JournalFibrinolysis and Proteolysis
Issue numberSUPPL. 3
StatePublished - 1997

ASJC Scopus subject areas

  • Hematology


Dive into the research topics of 'Targeting proteolysis to non-physiological protein substrates'. Together they form a unique fingerprint.

Cite this