Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site.
The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-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 alpha 1(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.(ABSTRACT TRUNCATED AT 250 WORDS)
Fields
G B
GB
Department of Chemistry, Florida State University, Tallahassee 32306.
Netzel-Arnett
S J
SJ
Windsor
L J
LJ
Engler
J A
JA
Birkedal-Hansen
H
H
Van Wart
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002364:Caseins; D009842:Oligopeptides; D005780:Gelatin; D003094:Collagen; D003012:Microbial Collagenase