Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes

Role of fibronectin-like domain and active site of gelatinase B

Thomas J. O'Farrell, Tayebeh Pourmotabbed

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu397, Ala406, Asp410, and Pro415, in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu397 and Ala406 are important for the general proteolytic activity of the enzyme, whereas Asp410 and Pro415 specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.

Original languageEnglish (US)
Pages (from-to)27964-27972
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number36
DOIs
StatePublished - Sep 8 2000

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Matrix Metalloproteinase 8
Matrix Metalloproteinase 9
Collagen Type V
Fibronectins
Gelatinases
Catalytic Domain
Enzymes
Substrate Specificity
Matrix Metalloproteinases
Amino Acids
Pathologic Processes
Substrates
Gelatin
Catalysis
Extracellular Matrix
Zinc
Tumors
Digestion
Peptide Hydrolases
Substitution reactions

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes: Role of fibronectin-like domain and active site of gelatinase B",
abstract = "Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu397, Ala406, Asp410, and Pro415, in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu397 and Ala406 are important for the general proteolytic activity of the enzyme, whereas Asp410 and Pro415 specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.",
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AB - Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu397, Ala406, Asp410, and Pro415, in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu397 and Ala406 are important for the general proteolytic activity of the enzyme, whereas Asp410 and Pro415 specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.

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