Year-end Sale % OFF 
Abstract
The collagen prolyl 4-hydroxylases (collagen P4Hs, EC 1.14.11.2) play a key role in the synthesis of the extracellular matrix. The vertebrate enzymes are alpha(2)beta(2) tetramers, the beta subunit being identical to protein disulfide isomerase (PDI). The main Caenorhabditis elegans collagen P4H form is an unusual PHY-1/PHY-2/(PDI)(2) mixed tetramer consisting of two types of catalytic alpha subunit, but the PHY-1 and PHY-2 polypeptides also form active PHY/PDI dimers. The lengths of peptide substrates have a major effect on their interaction with the P4H tetramers, the K(m) values decreasing markedly with increasing chain length. This phenomenon has been explained in terms of processive binding of the two catalytic subunits to long peptides. We determined here the K(m) values of a collagen P4H having two catalytic sites, the C. elegans mixed tetramer, and a form having only one such site, the PHY-1/PDI dimer, for peptides of varying lengths. All the K(m) values of the PHY-1/PDI dimer were found to be about 1.5-2.5 times those of the tetramer, but increasing peptide length led to identical decreases in the values of both enzyme forms. The K(m) for a nonhydroxylated collagen fragment with 33 -X-Y-Gly-triplets but only 11 -X-Pro-Gly-triplets was found to correspond to the number of the former rather than the latter. To study the individual roles of the two catalytic sites in a tetramer, we produced mutant PHY-1/PHY-2/(PDI)(2) tetramers in which binding of the Fe(2+) ion or 2-oxoglutarate to one of the two catalytic sites was prevented. The activities of the mutant tetramers decreased to markedly less than 50% of that of the wild type, being about 5-10% and 20-30% with the enzymes having one of the two Fe(2+)-binding sites or 2-oxoglutarate-binding sites inactivated, respectively, while the K(m) values for these cosubstrates or peptide substrates were not affected. Our data thus indicate that although collagen P4Hs do not act on peptide substrates by a processive mechanism, prevention of hydroxylation at one of the two catalytic sites in the tetramer impairs the function of the other catalytic site.
Highlights
The collagen prolyl 4-hydroxylases (P4Hs)1 catalyze the formation of 4-hydroxyproline in -X-Pro-Gly- sequences in collagens and more than 20 proteins with collagen-like domains [1,2,3,4,5]
Increasing Substrate Chain Length Reduces the Km Values of the PHY-1/PHY-2/(PDI-2)2 Tetramer and PHY-1/Human protein disulfide isomerase (PDI) Dimer —According to the hypothesis of processive action of the two peptide-binding sites in the collagen P4H tetramer, an enzyme with two such sites should be much more efficient in hydroxylating long peptide substrates than an enzyme with only one site, whereas the synergistic relation between the two sites should not be evident in the hydroxylation of short peptides [21, 22]
To study the suggested co-operation between the two peptide-binding sites in more detail, a recombinant C. elegans PHY-1/PHY-2/(PDI-2)2 tetramer, i.e. a P4H with two peptidebinding and catalytic sites, and the PHY-1/human PDI dimer, i.e. a P4H with one peptide-binding and one catalytic site, were produced in insect cells (Fig. 1) and their Km values for peptide substrates of four lengths determined using an activity assay based on the hydroxylation-coupled decarboxylation of 2-oxo[114C]glutarate
Summary
The collagen prolyl 4-hydroxylases (P4Hs) catalyze the formation of 4-hydroxyproline in -X-Pro-Gly- sequences in collagens and more than 20 proteins with collagen-like domains [1,2,3,4,5]. The Km values of the collagen P4Hs for peptide substrates decrease markedly with increasing chain length of the substrate [1,2,3] This finding has been explained by a processive mechanism of binding of long peptide substrates [2, 21, 22]. According to this model the enzyme-substrate complex is formed at one catalytic site upon. Lower Km values with increasing substrate chain length were observed in the case of both the enzyme tetramer and the dimer, the results suggesting that these occur on account of the higher affinity of the peptidesubstrate-binding domain for long peptides rather than through processive binding. The P4H activities of the mutant tetramers were markedly less than 50% of that of the wild type, indicating that the remaining single wild-type catalytic site in the mutants does not function entirely independently
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
