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)
Pages (from-to)51
Number of pages1
JournalFibrinolysis and Proteolysis
Volume11
Issue numberSUPPL. 3
StatePublished - 1997

Fingerprint

Proteolysis
Proteins
Peptides
Micrococcal Nuclease
Serine Proteases
Trypsin
Peptide Hydrolases

ASJC Scopus subject areas

  • Hematology

Cite this

Corey, D. R., Coombs, G. S., Bergstrom, R. C., & Madison, E. L. (1997). Targeting proteolysis to non-physiological protein substrates. Fibrinolysis and Proteolysis, 11(SUPPL. 3), 51.

Targeting proteolysis to non-physiological protein substrates. / Corey, David R.; Coombs, Gary S.; Bergstrom, Robert C.; Madison, Edwin L.

In: Fibrinolysis and Proteolysis, Vol. 11, No. SUPPL. 3, 1997, p. 51.

Research output: Contribution to journalArticle

Corey, DR, Coombs, GS, Bergstrom, RC & Madison, EL 1997, 'Targeting proteolysis to non-physiological protein substrates', Fibrinolysis and Proteolysis, vol. 11, no. SUPPL. 3, pp. 51.
Corey, David R. ; Coombs, Gary S. ; Bergstrom, Robert C. ; Madison, Edwin L. / Targeting proteolysis to non-physiological protein substrates. In: Fibrinolysis and Proteolysis. 1997 ; Vol. 11, No. SUPPL. 3. pp. 51.
@article{ac1ad8ec9d344666bb1f2df696107484,
title = "Targeting proteolysis to non-physiological protein substrates",
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.",
author = "Corey, {David R.} and Coombs, {Gary S.} and Bergstrom, {Robert C.} and Madison, {Edwin L.}",
year = "1997",
language = "English (US)",
volume = "11",
pages = "51",
journal = "Fibrinolysis and Proteolysis",
issn = "1369-0191",
publisher = "Churchill Livingstone",
number = "SUPPL. 3",

}

TY - JOUR

T1 - Targeting proteolysis to non-physiological protein substrates

AU - Corey, David R.

AU - Coombs, Gary S.

AU - Bergstrom, Robert C.

AU - Madison, Edwin L.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33846699807&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846699807&partnerID=8YFLogxK

M3 - Article

VL - 11

SP - 51

JO - Fibrinolysis and Proteolysis

JF - Fibrinolysis and Proteolysis

SN - 1369-0191

IS - SUPPL. 3

ER -