Proteolytic Activities of Human Fibroblast Collagenase

Hydrolysis of a Broad Range of Substrates at a Single Active Site

Gregg B. Fields, Sarah J. Netzel-Arnett, L. Windsor, Jeffrey A. Engler, Henning Birkedal-Hansen, Harold E. Van Wart

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat α 1(I) gelatin, β-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the αl(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site. On the basis of these kinetic experiments, it is concluded that HFC has a single active site that is capable of hydrolyzing a much wider variety of natural and synthetic substrates than previously believed.

Original languageEnglish (US)
Pages (from-to)6670-6677
Number of pages8
JournalBiochemistry
Volume29
Issue number28
DOIs
StatePublished - Jul 1 1990
Externally publishedYes

Fingerprint

Matrix Metalloproteinase 8
Human Activities
Hydrolysis
Catalytic Domain
Substrates
Gelatin
Collagen Type I
Caseins
prolyl-leucyl-glycine
Rats
Oligopeptides
Peptides
Collagenases
Fibroblasts
Kinetic parameters
Escherichia coli
Purification
Conformations
Assays
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fields, G. B., Netzel-Arnett, S. J., Windsor, L., Engler, J. A., Birkedal-Hansen, H., & Van Wart, H. E. (1990). Proteolytic Activities of Human Fibroblast Collagenase: Hydrolysis of a Broad Range of Substrates at a Single Active Site. Biochemistry, 29(28), 6670-6677. https://doi.org/10.1021/bi00480a017

Proteolytic Activities of Human Fibroblast Collagenase : Hydrolysis of a Broad Range of Substrates at a Single Active Site. / Fields, Gregg B.; Netzel-Arnett, Sarah J.; Windsor, L.; Engler, Jeffrey A.; Birkedal-Hansen, Henning; Van Wart, Harold E.

In: Biochemistry, Vol. 29, No. 28, 01.07.1990, p. 6670-6677.

Research output: Contribution to journalArticle

Fields, GB, Netzel-Arnett, SJ, Windsor, L, Engler, JA, Birkedal-Hansen, H & Van Wart, HE 1990, 'Proteolytic Activities of Human Fibroblast Collagenase: Hydrolysis of a Broad Range of Substrates at a Single Active Site', Biochemistry, vol. 29, no. 28, pp. 6670-6677. https://doi.org/10.1021/bi00480a017
Fields, Gregg B. ; Netzel-Arnett, Sarah J. ; Windsor, L. ; Engler, Jeffrey A. ; Birkedal-Hansen, Henning ; Van Wart, Harold E. / Proteolytic Activities of Human Fibroblast Collagenase : Hydrolysis of a Broad Range of Substrates at a Single Active Site. In: Biochemistry. 1990 ; Vol. 29, No. 28. pp. 6670-6677.
@article{21cfcacb9f6741d788d643df33583793,
title = "Proteolytic Activities of Human Fibroblast Collagenase: Hydrolysis of a Broad Range of Substrates at a Single Active Site",
abstract = "The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat α 1(I) gelatin, β-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the αl(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site. On the basis of these kinetic experiments, it is concluded that HFC has a single active site that is capable of hydrolyzing a much wider variety of natural and synthetic substrates than previously believed.",
author = "Fields, {Gregg B.} and Netzel-Arnett, {Sarah J.} and L. Windsor and Engler, {Jeffrey A.} and Henning Birkedal-Hansen and {Van Wart}, {Harold E.}",
year = "1990",
month = "7",
day = "1",
doi = "10.1021/bi00480a017",
language = "English (US)",
volume = "29",
pages = "6670--6677",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "28",

}

TY - JOUR

T1 - Proteolytic Activities of Human Fibroblast Collagenase

T2 - Hydrolysis of a Broad Range of Substrates at a Single Active Site

AU - Fields, Gregg B.

AU - Netzel-Arnett, Sarah J.

AU - Windsor, L.

AU - Engler, Jeffrey A.

AU - Birkedal-Hansen, Henning

AU - Van Wart, Harold E.

PY - 1990/7/1

Y1 - 1990/7/1

N2 - The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat α 1(I) gelatin, β-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the αl(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site. On the basis of these kinetic experiments, it is concluded that HFC has a single active site that is capable of hydrolyzing a much wider variety of natural and synthetic substrates than previously believed.

AB - The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat α 1(I) gelatin, β-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the αl(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site. On the basis of these kinetic experiments, it is concluded that HFC has a single active site that is capable of hydrolyzing a much wider variety of natural and synthetic substrates than previously believed.

UR - http://www.scopus.com/inward/record.url?scp=0025292351&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025292351&partnerID=8YFLogxK

U2 - 10.1021/bi00480a017

DO - 10.1021/bi00480a017

M3 - Article

VL - 29

SP - 6670

EP - 6677

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 28

ER -