Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation.
In agreement with others (Myllylä, R., Kuutti-Savolainen, E.-R. and Kivirikko, K.I. (1978) Biochem. Biophys. Res. Commun. 83, 441-448), it was found that, in the absence of ascorbate, prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) catalyses the hydroxylation of peptidyl proline, stoicheiometrically coupled to the oxidative decarboxylation of 2-oxoglutarate, at a high initial rate. Under these conditions the enzyme becomes inactivated by at least 90% within 1 min in the presence of 400 microM 2-oxoglutarate, in the presence or absence of the peptide substrate (Pro-Pro-Gly)10. The enzyme can be partly reactivated by ascorbate, but not by Fe2+. Addition of a stoicheiometric amount of iron to the enzyme gives rise to a small EPR signal at g = 4.3, which is typical of a high-spin d 5 ion in a rhombic environment. After subsequent incubation for 30 s at 37 degrees C in the presence of 2-oxoglutarate, the amplitude of the EPR signal at g = 4.3 increases 3-4-fold and corresponds to virtually all of the iron added. In addition, an EPR signal at g = 2.0 is formed under these conditions. The signal at g = 4.3 decreases after subsequent addition of ascorbate. It is concluded that in the presence of 2-oxoglutarate enzyme-bound Fe2+ is rapidly converted to Fe3+, leading to inactivation of the enzyme. Enzyme-bound Fe3+ can be reduced again by ascorbate, thus reactivating the enzyme, or, in the absence of 2-oxoglutarate, by Fe2+.
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D002642:Chick Embryo; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000494:pharmacology; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011392:Proline / Q000378:metabolism
D007656:Ketoglutaric Acids; D011392:Proline; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D001205:Ascorbic Acid