Structural properties of porcine intestine acylpeptide hydrolase.
The acylpeptide hydrolase of porcine intestinal mucosa (pi-APH) is a serine peptidase belonging to the prolyl oligopeptidase family. The enzyme catalyzes the release of N-terminal acylamino acids, especially acetylamino acids, from acetylpeptides. pi-APH is an homotetramer of approximately 300 kDa. We report the loss of the native tetrameric structure of pi-APH upon citraconylation and the process was reversed at acidic pH, indicating that the subunits were noncovalently bound. Determination of free cysteines in combination with peptide mapping suggested the involvement of all cysteines in disulfide bridges. Two structural domains were identified based on the three-dimensional model of pi-APH monomer: a beta-propeller fold in the N-terminal sequence (113-455) and an alpha/beta hydrolase fold corresponding to the C-terminal catalytic domain (469-732). Preferential cleavage sites for limited proteolysis with trypsin occurred within the beta-propeller domain, in agreement with the three-dimensional model. The putative role of this domain in the specificity mechanism of APH enzymes is also discussed.
Durand
Anne
A
Institut Méditerranéen de Recherche en Nutrition, UMR Université Aix-Marseille III-INRA 1111, Faculté des Sciences et Techniques de St-Jérôme, Case 342, 13397 Marseille Cedex 20, France.
Villard
Claude
C
Giardina
Thierry
T
Perrier
Josette
J
Juge
Nathalie
N
Puigserver
Antoine
A
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000201:enzymology*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014357:Trypsin
D010446:Peptide Fragments; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase; D014357:Trypsin; D003545:Cysteine