Abstract
Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.