title: The purification, characterization and analysis of primary and secondary-structure of prolyl oligopeptidase from human lymphocytes. Evidence that the enzyme belongs to the alpha/beta hydrolase fold family.
abstract: Prolyl oligopeptidase was isolated and purified to homogeneity from human lymphocytes, yielding a specific activity of 7780 mU/mg. The molecular mass using size-exclusion chromatography matches the 76 kDa obtained by SDS/PAGE. This provides evidence that prolyl oligopeptidase is a monomer. The isoelectric point is 4.8 as judged by isoelectric focusing in free solution. Di-isopropyl fluorophosphate and phenylmethylsulphonyl fluoride completely abolish the activity, classifying the enzyme as a serine proteinase. The inhibition by p-chloromercuribenzoic acid indicates the importance of a free sulfhydryl group near the active-site. alpha 1-Casein and ornithine decarboxylase, two proteins containing a PEST sequence, inhibit prolyl oligopeptidase, but were not hydrolyzed. This demonstrates that prolyl oligopeptidase is not participating in the metabolism of proteins according to a PEST-dependent pathway. alpha 1-Antitrypsin partially inhibits the enzyme but in contrast, aprotinin does not. Its inability to cleave corticotropin-releasing factor, ubiquitin, albumin and aprotinin, together with the hydrolysis of bradykinin between Pro7-Arg8 confirms the affinity of prolyl oligopeptidase for small peptides. Multiple sequence alignment does not reveal any similarity with proteases of known tertiary structure. Secondary-structure prediction displays striking similarity with dipeptidyl peptidase IV and acylaminoacyl peptidase. Two characteristic features of the members of the prolyl oligopeptidase family of serine proteases are high-lighted: the linear arrangement of the catalytic triad is nucleophile-acid-base and the proteolytic cleavage releasing the catalytically active C-terminal region of around 500 amino acids from the N-terminal sequence. Secondary structure prediction and comparison of the active-site of serine proteinases with known three-dimensional coordinates prove that Asp641 is the third member of the catalytic triad. The secondary structural organization of the protease domain of prolyl oligopeptidase is in accordance with the alpha/beta hydrolase fold.
authors.affiliation: Laboratory for Medical Biochemistry, University of Antwerp, Antwerpen, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: Goossens
authors.forename: I
authors.initials: I
authors.lastname: De Meester
authors.forename: G
authors.initials: G
authors.lastname: Vanhoof
authors.forename: D
authors.initials: D
authors.lastname: Hendriks
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: S
authors.initials: S
authors.lastname: Scharpé
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases