Note. The novel amino acid sequence data published here have been submitted to the Genbank sequence data bank and are available under accession number X74496.
The Purification, Characterization and Analysis of Primary and Secondary-Structure of Prolyl Oligopeptidase from Human Lymphocytes
Evidence that the Enzyme Belongs to the α/β Hydrolase Fold Family
Article first published online: 7 SEP 2004
European Journal of Biochemistry
Volume 233, Issue 2, pages 432–441, October 1995
How to Cite
Goossens, F., De Meester, I., Vanhoof, G., Hendriks, D., Vriend, G. and Scharpé, S. (1995), The Purification, Characterization and Analysis of Primary and Secondary-Structure of Prolyl Oligopeptidase from Human Lymphocytes. European Journal of Biochemistry, 233: 432–441. doi: 10.1111/j.1432-1033.1995.432_2.x
- Issue published online: 7 SEP 2004
- Article first published online: 7 SEP 2004
- (Received 30 March/11 July 1995) – EJB 95 0505/4
- prolyl oligopeptidase;
- sequence analysis;
- PEST hypothesis;
- α/β hydrolase fold
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. α1-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. α1-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 highlighted: 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 α/β hydrolase fold.