Mutational analysis of the thyrotropin-releasing hormone-degrading ectoenzyme. similarities and differences with other members of the M1 family of aminopeptidases and thermolysin.
Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE) is a TRH-specific peptidase which catalyzes the inactivation of the peptidergic signal substance TRH. As indicated by sequence alignment, TRH-DE and the other members of the M1 family of aminopeptidases have a distinct set of conserved amino acid residues in common. By replacing amino acid residues that are putatively involved in catalysis, we could demonstrate that the enzymatic activities of the mutants E408D, E442D, E464Q, E464D, Y528F, H507R, and H507F are dramatically decreased, essentially due to the changes of V(max). The mutant enzymes E408Q and E442Q are inactive, whereas the specific enzymatic activity of the mutants R488Q, R488A, and Y554F are similar to that of the wild-type enzyme. These data strongly suggest that E408, E442, Y528, and H507 are involved in the catalytic process of TRH-DE while E464 presumably represents the third zinc-coordinating residue and may be equivalent to E166 in thermolysin. In contrast, amino acid residues R488 and Y554 seem not to be involved in the catalytic mechanism of TRH-DE.
Papadopoulos
T
T
Max-Planck-Institut für Experimentelle Endokrinologie, 30603 Hannover, Germany.
Kelly
J A
JA
Bauer
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000145:classification / Q000494:pharmacology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013820:Thermolysin / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014162:Transfection
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013973:Thyrotropin-Releasing Hormone; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D013820:Thermolysin; D011761:Pyrrolidonecarboxylic Acid