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Neuropeptide substrate specificity in thimet oligopeptidase is conferred by glycine residues residing in a loop region
Author(s) -
Srikanthan Meera A.,
Song M. M.,
Bruce L. A.,
Kua L. H.,
Pabon A.,
Glucksman M. J.,
Sigman J. A.,
Elmore D. E.,
Wolfson A. J.
Publication year - 2007
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.21.6.a1014-d
Subject(s) - chemistry , oligopeptidase , mutant , stereochemistry , substrate (aquarium) , residue (chemistry) , biochemistry , enzyme , biology , ecology , gene
Thimet oligopeptidase is a metallozenzyme with broad specificity for small bioactive neuropeptides. We have previously shown that flexibility in a loop region comprised of residues 599–611 is essential for substrate binding and catalysis. By mutating Gly residues in the loop to Ala, individually and in combination, we now identify G603 as the position that determines substrate specificity. A form of the enzyme with Ala in place of G603 has a strong preference for a 5‐residue substrate, compared to wild type and other engineered forms that have preference for a 9‐residue derivative of the natural substrate bradykinin. In contrast, the specificity of G604A is unchanged with respect to wild type. All double mutants containing A603 show the same preference as that with the single mutation. This preference appears to be associated with rigidity in the loop and a tighter conformation conferred by the Ala substitution. Inclusion of an osmolyte that promotes protein folding has little effect on G603A but leads to enhanced activity of wild type and other Ala‐substituted forms towards the 5‐residue substrate. Computer simulations support the idea that the Ala insertion in the loop leads to less flexibility in that region of the enzyme. We surmise that flexibility in the loop allows the enzyme to bind a variety of neuropeptide substrates, which it can specifically process to products that modulate physiological processes in neurons.