prolyl 4-hydroxylases (EC 188.8.131.52
, procollagen-proline, 2-oxoglutarate, 4-dioxygenases) reside in the lumen of the endoplasmic reticulum, where they catalyze the hydroxylation of proline residues in X-Pro-Gly sequences in collagens and the collagenous sequences of other proteins.
The vertebrate collagen prolyl 4-hydroxylases are tetrameric enzymes composed of two α subunits and two β subunits, with a total molecular weight of 240 kDa. The β subunit
is identical to protein disulphide isomerase (PDI, EC 184.108.40.206
that is a enzyme located within the lumen of the endoplasmic reticulum, where it catalyzes the formation and rearrangement of disulphide bonds in the biosynthesis of various secretory and cell surface proteins, including collagens), the α subunit being responsible for the catalytic activity of the enzyme tetramer.
The type I collagen prolyl 4-hydroxylase is the main form in most cell types and tissues, while type II has been shown to be a major form in chondrocytes, osteoblasts, endothelial cells and cells in epithelial structures.
The expression of the type III collagen prolyl 4-hydroxylase seems to be more restricted: only in the placenta, adult liver and foetal skin.
All the α subunits are synthesized in a form containing a signal peptide, the size of which varies between 16 and 21 amino acids. Prolyl 4-hydroxylase, 2010
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The sequences of the α subunit isoforms are highly conserved between species: the human α(III) sequence, for example, shows 91% and 94% identity to the corresponding rat and mouse sequences, respectively.
There are five conserved cysteine residues in the human α(I), α(II) and α(III) subunits: the second and third conserved cysteines form one intrachain disulphide bond and the fourth and fifth cysteines another; these bonds being essential for maintaining the tertiary structure of the α subunit needed for tetramer assembly. No interchain disulphide bonds exist between the subunits within the tetramer.
The gene for the human α(I) subunit consists of 16 exons and is located on chromosome 10, while the genes for the α(II) and α(III) subunits are located on chromosomes 5 and 11, respectively.
β subunit of the human type I collagen prolyl 4-hydroxylase is known to have protein disulphide isomerase activity even when present in the collagen prolyl 4-hydroxylase tetramer. This PDI activity is not likely to be part of the hydroxylation mechanism, but a monoclonal antibody to PDI has been shown to partially inhibit the enzyme activity, indicating that some region of the PDI subunit may be located close to the catalytic sites. The main part of PDI is composed of four thioredoxin modules, two of them being catalytically active and the other two inactive.
The C terminus of the PDI polypeptide contains the -Lys-Asp-Glu-Leu sequence, which is both necessary and sufficient for the retention of a polypeptide within the lumen of the endoplasmic reticulum.
If the collagen prolyl 4-hydroxylase tetramer is somehow dissociated, the α subunit immediately forms insoluble aggregates: when an α subunit is expressed alone, without the PDI
subunit, it forms insoluble aggregates that have no prolyl 4-hydroxylase activity.
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The prolyl 4-hydroxylases belong to the group of 2-oxoglutarate and non-heme-Fe(II)-dependent dioxygenases, which all require Fe2+ , 2-oxoglutarate, O2 and ascorbate
for catalytic activity.
Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase, 1984.
Ascorbate may be required to prevent oxidation of the enzyme-bound Fe2+, and possibly some other groups on the enzyme molecule, by a side reaction during some, but not the majority, of the catalytic cycles.
This side reaction is probably the uncoupled decarboxylation of 2-oxoglutarate. In the complete reaction the decarboxylation of 2-oxoglutarate probably leads to the formation of a ferryl ion that acts as the active intermediate in oxygen transfer and hydroxylates the peptidebound proline or lysine residue. In the uncoupled reaction, however, the reactive iron-oxo complex is probably converted to Fe3+ and OH‘, and the Fe3+ ion remains bound to the active site, making the enzyme unavailable for new catalytic cycles. The function of ascorbate appears to be to reactivate such an enzyme by reducing the enzyme-bound Fe3+ .
The prolyl 4-hydroxylases also catalyze an uncoupled decarboxylation of 2-oxoglutarate, that is, decarboxylation without subsequent hydroxylation, even in the presence of a peptide substrate.
The main function of ascorbate thus seems to be to reactivate the enzyme after such uncoupled cycles, having a role as an alternative oxygen acceptor; in the absence of ascorbate, prolyl 4-hydroxylase is rapidly inactivated by self-oxidation.
Schematic representation of the reaction of P4H
According to the current model for the catalytic site and reaction mechanism, the Fe2+ is located in a supposedly highly hydrophobic pocket coordinated with the enzyme by three side-chains, the residues being histidines 412 and 483 and aspartate 414 in the human α(I) subunit. Three distinct subsites participate in the binding of 2-oxoglutarate: subsite I, lysine residue 493 in the human α(I) subunit, which ionically binds the C5
carboxyl group of the 2-oxoglutarate, subsite II consisting of two cis29 positioned coordination sites of the enzyme-bound Fe2+, which is chelated by the C1-C2 moiety, and subsite III, which involves a hydrophobic binding site in the C3-C4 region of the cosubstrate.
During the first half of the hydroxylation reaction, molecular oxygen is bound and 2-oxoglutarate is decarboxylated to succinate; at the same time a highly reactive iron-oxo complex, a ferryl ion, is formed. This acts as the active intermediate in oxygen transfer, hydroxylating the proline residue in the peptide substrate in the second half of the hydroxylation reaction.
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Ascorbate is required to prevent oxidation of the enzyme-bound Fe2+ , and possibly some other groups on the enzyme molecule, during some catalytic cycles, but not the majority.
The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. The prolyl 4-hydroxylases only hydroxylate prolines in peptide linkages, not free proline.
The minimum sequence requirement for hydroxylation by the vertebrate collagen prolyl 4-hydroxylases is a X-Pro-Gly triplet;
The affinity of collagen
prolyl 4-hydroxylases for their peptide substrates is further affected by the chain lenght of the peptide. Poly(L-proline) is an effective competitive inhibitor of the vertebrate type I collagen prolyl 4-hydroxylase.
The level of collagen prolyl 4-hydroxylase activity in cells and tissues usually correlates with the rate of collagen synthesis. The subunits may form a tetramer, but this will rapidly dissociate back to its subunits in the absence of collagen synthesis. The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied.
Purified prolyl 4-hydroxylase is able to accept a number of ascorbate analogues instead of the physiological substrate, if the two hydroxy groups participating in the redox reaction remain unchanged.
Compounds with substitutions at the ring-hydroxy groups neither support the enzymic reaction nor do they act as inhibitors.
This finding suggests a direct participation of the ring hydroxy groups in the binding of this co-substrate. By contrast, hydroxy groups located in the side chain of ascorbate are neither necessary for binding of this co-substrate to the enzyme's active site nor for its transport through the microsomal membrane.
The prolyl 4-hydroxylases
that catalyze hydroxylation of the transcription factor called hypoxia-inducible factor HIF
α comprise a novel cytoplasmic enzyme family distinct from the collagen prolyl 4-hydroxylases.
The three HIF
prolyl 4-hydroxylases require the same cosubstrates as the collagen prolyl 4-hydroxylases, but their Km values for O2 are much higher than that of the type I collagen prolyl 4-hydroxylase, being just slightly above the atmospheric oxygen concentration, which is in keeping with their function as effective oxygen sensors.