Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

Abstract

Pyroglutamyl peptidase II (PPII), a highly specific membranebound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (<Glu-His-Pro-NH 2 ), inactivating the peptide in the extracellular space. PPII and enzymes with distinct specificities such as neutral aminopeptidase (APN), belong to the M1 metallopeptidase family. M1 aminopeptidases recognize the N-terminal amino group of substrates or inhibitors through hydrogen-bonding to two conserved residues (Gln-213 and exopeptidase motif Glu-355 in human APN), whereas interactions involved in recognition of pyroglutamyl residue by PPII are unknown. In rat PPII, the conserved exopeptidase residue is Glu-408, whereas the other one is Ser-269. Given that variations in M1 peptidase specificity are likely due to changes in the catalytic region, we constructed three-dimensional models for the catalytic domains of PPII and APN. The models showed a salt bridge interaction between PPII-Glu-408 and PPII-Lys-463, whereas the equivalent APN-Glu-355 did not participate in a salt bridge. Docking of thyrotropin-releasing hormone in PPII model suggested that the pyroglutamyl residue interacted with PPII-Ser-269. According to our models, PPII-S269Q and -K463N mutations should leave Glu-408 in a physicochemical context similar to that found in M1 aminopeptidases; alternatively, PPII-S269E replacement might be sufficient to transform PPII into an aminopeptidase. These hypotheses were supported by site-directed mutagenesis; the mutants lost omegapeptidase but displayed alanyl-aminopeptidase activity. In conclusion, recognition of a substrate without an N-terminal charge requires neutralization of the aminopeptidase anionic binding site; furthermore, shortening of side chain at PPII-269 position is required for adjustment to the pyroglutamyl residue.

The Thyrotropin-Releasing Hormone-Degrading Ectoenzyme, a Therapeutic Target?

“…This agrees with evidence that Glp-Phe-Pro-NH 2 is a substrate of bovine brain TRH-DE (Kelly et al, 1997;Gallagher and O'Connor, 1998). The structural determinants of TRH-DE narrow specificity have been partially clarified; two amino-acid substitutions in the catalytic domain are critical and form a conserved signature that distinguishes it from other M1 family members (Chavez-Gutieŕrez et al, 2006).…”

“…Trhde gene sequences and enzymatic activities have been detected in mammals, including activity in human serum and cerebrospinal fluid (Prasad and Jayaraman, 1986;Bundgaard and Møss, 1990;Charli et al, 1998). The gene is also detected in other vertebrate classes (Schomburg et al, 1999;Chavez-Gutieŕrez et al, 2006). TRH-DE is not an essential gene in mice as TRH-DE KO animals are healthy, reproduce normally and their metabolic parameters are normal when bred in standard conditions (Tang et al, 2010).…”

“…TRH-DE is a member of the M1 family of zinc-aminopeptidases, a family of 12 members (in humans) that includes aminopeptidases that have a wider specificity than TRH-DE, including aminopeptidase N (the closest relative of TRH-DE), which preferentially hydrolyses neutral aminoacids from the N-terminus (Sjöström et al, 2002). Although TRH-DE 3D structure is still unknown, analogy with other members of the family suggests it is a type II cell surface peptidase with a small intracellular domain, a single transmembrane domain, and a large extracellular part with a flexible stem followed by a catalytic domain, and an additional domain separated by a flexible loop (Chavez-Gutieŕrez et al, 2006). Topology of the domains is consistent with biochemical evidence that demonstrated TRH-DE is an ectoenzyme (Charli et al, 1988), making it a prime candidate for primary hydrolysis of TRH in the extracellular space.…”

Crystal structures of pyrrolidone-carboxylate peptidase I from Deinococcus radiodurans reveal the mechanism of L-pyroglutamate recognition

“…Unfortunately, the structure of PCP II is not available from any organism. In the absence of the crystal structure of PCP II, a possible mechanism for pG recognition has been proposed based on a model built by homology modeling using human leukotriene A4 hydrolase as a template (Chá vez-Gutié rrez et al, 2006).…”