University of Oulu

Collagen prolyl 4-hydroxylase : characterization of a novel vertebrate isoenzyme and the main Caenorhabditis elegans enzyme forms, and effect of inactivation of one of the two catalytic sites in the enzyme tetramer

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Author: Kukkola, Liisa1,2,3
Organizations: 1University of Oulu, Collagen Research Unit
2University of Oulu, Biocenter Oulu
3University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
Format: ebook
Version: published version
Access: open
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Language: English
Published: 2003
Publish Date: 2003-12-05
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium L101 of the Department of Medical Biochemistry and Molecular Biology, on December 5th, 2003, at 10 a.m.
Reviewer: Professor Leena Brückner-Tuderman
Docent Merja Perälä


Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers.

This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA.

A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens.

The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

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Series: Acta Universitatis Ouluensis. D, Medica
ISSN-E: 1796-2234
ISBN: 951-42-7203-X
ISBN Print: 951-42-7202-1
Issue: 764
Copyright information: © University of Oulu, 2003. This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.