Raili Myllylä

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.

The kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double‐reciprocal plots for all possible pairs of Fe 2+ , 2‐oxoglutarate, O 2 and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concentration was varied at different fixed 2‐oxoglutarate concentrations in the presence of saturating O 2 concentration. Poly( L ‐proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe 2+ and 2‐oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe 2‐ and 2‐oxoglutarate. Succinate, CO 2 and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2‐oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent K m and K s values for the substrates and K i values for the inhibitors are given. These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe 2+ , 2‐oxoglutarate, O 2 and the polypeptide substrate to the enzyme in this order, the binding of Fe 2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly( L ‐proline) under certain conditions, forming dead‐end complexes. The products are released only after the hydroxylation, possibly in the order: the hydroxylated polypeptide, CO 2 and succinate. Ascorbate may react either with enzyme·Fe before the release of Fe 2+ or with free enzyme before the binding of Fe 2+ , but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2‐oxoglutarate dioxygenases.

The co‐substrate requirements of prolyl hydroxylase were studied with pure enzyme from chick embryos. No hydroxylation occurred without added Fe 2+ , indicating that the enzyme does not retain iron sufficiently to catalyze any reaction. Zn 2+ was an effective competitive inhibitor with respect to Fe 2+ , but was noncompetitive with respect to the polypeptide substrate and 2‐oxoglutarate, suggesting that it replaced iron in the active site of the enzyme. The enzyme catalyzed the uncoupled decarboxylation of 2‐oxoglutarate at a rate of about 4 mol CO 2 formed (mol enzyme) −1 min −1 in the presence of Fe 2+ , O 2 , and ascorbate but in the absence of the polypeptide substrate. This rate was about 1/80 of that observed in the presence of the substrate. Several compounds inhibited the enzyme competitively with respect to 2‐oxoglutarate but non‐competitively with respect to Fe 2+ . It seems that these two co‐substrates become bound at separate sites on the enzyme, and additional data suggested that these are distinct from the binding site of the polypeptide substrate. The reaction was completely dependent on O 2 . Nitroblue tetrazolium was a competitive inhibitor with respect to O 2 , but noncompetitive with respect to the polypeptide substrate and all other co‐substrates. Epinehrine also inhibited the enzyme, but this inhibition was competitive with respect to Fe 2+ . The results suggest that nitroblue tetrazolium consumed an activated form of oxygen, whereas epinephrine acted primarily by binding Fe 2+ . The reaction was completely dependent on ascorbate, and in contrast to previous data, this could not be significantly replaced by tetrahydrofolic acid or dithiothreitol. Dehydroascorbate replaced ascorbate in the presence of dithiothreitol but not in its absence. The results also indicate that ascorbate is not stoichiometrically consumed during the reaction.