An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase

L. Windsor, H. Birkedal-Hansen, B. Birkedal-Hansen, J. A. Engler

Research output: Contribution to journalArticle

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Abstract

The cDNA that encodes the proenzyme form of human fibroblast collagenase has been expressed in Escherichia coli. It has been shown by a number of criteria to be functionally identical with the enzyme isolated from human sources. Mutations of each of three cysteine residues found in procollagenase were constructed by site-directed mutagenesis of the cDNA. The relative activities of these mutants were compared to the wild-type enzyme. All of the mutants retained proteolytic activity, but not necessarily on collagen. Mutations that interfere with the formation of the sulfhydryl bridge in the carboxy-terminal domain in some cases increased and in other cases decreased the rate of casein cleavage. On the basis of extensive autolysis within E. coli of a mutant with a replacement of cysteine-73, the procollagenase molecule produced appeared to be either spontaneously active or perhaps more susceptible to autolytic activation, despite the continued presence of the propeptide. Experiments designed to capture the active forms of the mutant by use of the irreversible inhibitor α2-macroglobulin showed that some degree of latency still persisted in the autolytic mutant. These findings suggest that the cysteine at position 73 is important for maintaining the proenzyme in an inactive state but that the maintenance of latency in MMPs may be a complex process, involving a number of interactions between the propeptide domain and the remainder of the collagenase molecule.

Original languageEnglish (US)
Pages (from-to)641-647
Number of pages7
JournalBiochemistry
Volume30
Issue number3
StatePublished - 1991
Externally publishedYes

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Matrix Metalloproteinase 8
Cysteine
Enzyme Precursors
Maintenance
Escherichia coli
Complementary DNA
Autolysis
Macroglobulins
Mutagenesis
Mutation
Molecules
Collagenases
Enzymes
Site-Directed Mutagenesis
Caseins
Matrix Metalloproteinases
Collagen
Chemical activation
Experiments
procollagenase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Windsor, L., Birkedal-Hansen, H., Birkedal-Hansen, B., & Engler, J. A. (1991). An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase. Biochemistry, 30(3), 641-647.

An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase. / Windsor, L.; Birkedal-Hansen, H.; Birkedal-Hansen, B.; Engler, J. A.

In: Biochemistry, Vol. 30, No. 3, 1991, p. 641-647.

Research output: Contribution to journalArticle

Windsor, L, Birkedal-Hansen, H, Birkedal-Hansen, B & Engler, JA 1991, 'An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase', Biochemistry, vol. 30, no. 3, pp. 641-647.
Windsor, L. ; Birkedal-Hansen, H. ; Birkedal-Hansen, B. ; Engler, J. A. / An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase. In: Biochemistry. 1991 ; Vol. 30, No. 3. pp. 641-647.
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