| 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 - Goossens_1995_Eur.J.Biochem_233_432 |
| Author(s) : Goossens F , De Meester I , Vanhoof G , Hendriks D , Vriend G , Scharpe S |
| Ref : European Journal of Biochemistry , 233 :432 , 1995 |
| 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. |
| ESTHER : Goossens_1995_Eur.J.Biochem_233_432 |
| PubMedSearch : Goossens_1995_Eur.J.Biochem_233_432 |
| PubMedID: 7588785 |
| Gene_locus related to this paper: human-PREP |
| Gene_locus related to this paper: human-PREP |
Goossens F, De Meester I, Vanhoof G, Hendriks D, Vriend G, Scharpe S (1995)
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
European Journal of Biochemistry
233 :432
Goossens F, De Meester I, Vanhoof G, Hendriks D, Vriend G, Scharpe S (1995)
European Journal of Biochemistry
233 :432