Protein Engineering, Vol. 13, No. 6, 397-405,
June 2000
© 2000 Oxford University Press
Expression, characterization and structure determination of an active site mutant (Glu202–Gln) of mini-stromelysin-1
1 Department of Biochemistry and Molecular Genetics, 2 Oral Cancer Research Center and 3 Research Center in Oral Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA, 4 Department of Medicinal Chemistry, Victorian College of Pharmacy, Parkville, Vic. 3052, Australia, 5 Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, and 6 Department of Oral Biology, Indiana University, Indianapolis, IN 46202, USA
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 wild-type and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.
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