Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1

Darin L. Steele, O. El-Kabbani, P. Dunten, L. Jack Windsor, R. Ursula Kammlott, R. L. Crowther, C. Michoud, Jeffrey A. Engler, J. J. Birktoft

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) family of enzymes. The active site glutamic acid of the MMPs is conserved throughout the family and plays a pivotal role in the catalytic mechanism. The structural and functional consequences of a glutamate to glutamine substitution in the active site of stromelysin-1 were investigated in this study. In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activated by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue inhibitor of metalloproteinases, after cleavage of the propeptide with trypsin, A second construct containing the glutamine substitution but lacking the propeptide was also inactive in the proteolysis assays and capable of TIMP-1 binding. X-ray structures of the wild-type and mutant proteins complexed with the propeptide-based inhibitor Ro-26-2812 were solved and in both structures the inhibitor binds in an orientation the reverse of that of the propeptide in the pro-form of the enzyme. The inhibitor makes no specific interactions with the active site glutamate and a comparison of the wildtype and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.

Original languageEnglish (US)
Pages (from-to)397-405
Number of pages9
JournalProtein Engineering
Volume13
Issue number6
StatePublished - Jul 24 2000

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Matrix Metalloproteinase 3
Glutamine
Catalytic Domain
Substitution reactions
Enzymes
Glutamic Acid
Assays
Tissue Inhibitor of Metalloproteinase-1
Proteolysis
Matrix Metalloproteinases
Substrates
Peptides
Tissue Inhibitor of Metalloproteinases
Tissue
Proteins
X rays
Mutant Proteins
Gelatin
Acids
Trypsin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Steele, D. L., El-Kabbani, O., Dunten, P., Windsor, L. J., Kammlott, R. U., Crowther, R. L., ... Birktoft, J. J. (2000). Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1. Protein Engineering, 13(6), 397-405.

Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1. / Steele, Darin L.; El-Kabbani, O.; Dunten, P.; Windsor, L. Jack; Kammlott, R. Ursula; Crowther, R. L.; Michoud, C.; Engler, Jeffrey A.; Birktoft, J. J.

In: Protein Engineering, Vol. 13, No. 6, 24.07.2000, p. 397-405.

Research output: Contribution to journalArticle

Steele, DL, El-Kabbani, O, Dunten, P, Windsor, LJ, Kammlott, RU, Crowther, RL, Michoud, C, Engler, JA & Birktoft, JJ 2000, 'Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1', Protein Engineering, vol. 13, no. 6, pp. 397-405.
Steele, Darin L. ; El-Kabbani, O. ; Dunten, P. ; Windsor, L. Jack ; Kammlott, R. Ursula ; Crowther, R. L. ; Michoud, C. ; Engler, Jeffrey A. ; Birktoft, J. J. / Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1. In: Protein Engineering. 2000 ; Vol. 13, No. 6. pp. 397-405.
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