Mechanism of the Prolyl Hydroxylase Reaction

Abstract

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.