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-specif ic peptidase which catalyzes the inactivation of the peptidergic signal sub stance TRH. As indicated by sequence alignment, TRH-DE and the other member s of the M1 family of aminopeptidases have a distinct set of conserved amin o acid residues in common. By replacing amino acid residues that are putati vely involved in catalysis, we could demonstrate that the enzymatic activit ies 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 activ ity 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 ar e involved in the catalytic process of TRH-DE while E464 presumably represe nts the third zinc-coordinating residue and may be equivalent to E166 in th ermolysin. In contrast, amino acid residues R488 and Y554 seem not to be in volved in the catalytic mechanism of TRH-DE.