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Evidence for a conformational change in thimet oligopeptidase and the machinery behind it
Author(s) -
Scognamillo Lori A,
Sigman Jeffrey A.,
Glucksman Marc J.,
Pabon Amanda,
Wolfson Adele J.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.502.5
Subject(s) - chemistry , oligopeptidase , enzyme , biophysics , cleave , pterin , conformational change , substrate (aquarium) , biochemistry , amino acid , homology modeling , stereochemistry , biology , ecology , cofactor
The metalloenzyme thimet oligopeptidase (TOP) has been shown to cleave numerous structurally distinct physiological peptides all under 18 amino acids in length. Our previous data shows that conformational changes in the enzyme influences its substrate specificity. The data, supported by homology modeling of TOP to a substrate‐bound form of DcP carboxypeptidase, indicates that a hinge‐bend movement in TOP may be crucial to enzymatic activity and could provide a mechanism for TOP's size restriction. Using the homology model, we have identified several charged residues on the enzyme surface that line the entrance to the substrate‐binding crevice. We propose that these residues stabilize the closed form of the enzyme via ionic interactions. Quenched fluorescence‐based activity assays performed with the single and double mutants in which the charge on opposite sides of the cleft are reversed showed that these amino acids were crucial in maintaining enzymatic activity. Furthermore, chemical denaturation and fluorescence anisotropy reveal that the ability of the enzyme to form a closed structure may have been compromised. The data lends further support to the importance of hinge‐bend movements in TOP and supports a role for surface charges in stabilizing the closed form of the enzyme.