Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residues. Cys68 is involved in disulfide-linked dimerization.
Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein-protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.
Papadopoulos
T
T
Max-Planck-Institut für Experimentelle Endokrinologie, Hannover, Germany.
Heuer
H
H
Bauer
K
K
D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics*; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats
D017931:DNA Primers; D004220:Disulfides; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D003545:Cysteine; D011761:Pyrrolidonecarboxylic Acid