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 language||English (US)|
|Number of pages||1|
|Journal||Fibrinolysis and Proteolysis|
|Issue number||SUPPL. 3|
|State||Published - Dec 1 1997|
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