Targeting proteolysis to non-physiological protein substrates

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

Research output: Contribution to journalArticle

Abstract

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)
Number of pages1
JournalFibrinolysis and Proteolysis
Volume11
Issue numberSUPPL. 3
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Hematology

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