Abstract
Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 10788-10793 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 276 |
| Issue number | 14 |
| DOIs | |
| State | Published - Apr 6 2001 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Reaction kinetics of protease with substrate phage : Kinetic model developed using stromelysin. / Sharkov, Nikolai A.; Davis, Robyn M.; Reidhaar-Olson, John F.; Navre, Marc; Cai, Danying.
In: Journal of Biological Chemistry, Vol. 276, No. 14, 06.04.2001, p. 10788-10793.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Reaction kinetics of protease with substrate phage
T2 - Kinetic model developed using stromelysin
AU - Sharkov, Nikolai A.
AU - Davis, Robyn M.
AU - Reidhaar-Olson, John F.
AU - Navre, Marc
AU - Cai, Danying
PY - 2001/4/6
Y1 - 2001/4/6
N2 - Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.
AB - Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.
UR - http://www.scopus.com/inward/record.url?scp=0035815741&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035815741&partnerID=8YFLogxK
U2 - 10.1074/jbc.M011772200
DO - 10.1074/jbc.M011772200
M3 - Article
C2 - 11152696
AN - SCOPUS:0035815741
VL - 276
SP - 10788
EP - 10793
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 14
ER -