Counts D F, Cardinale G J, Udenfriend S
Proc Natl Acad Sci U S A. 1978 May;75(5):2145-9. doi: 10.1073/pnas.75.5.2145.
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.
脯氨酰羟化酶（脯氨酸、2-氧代戊二酸双加氧酶，EC 1.14.11.2）是一种混合功能氧化酶，它使肽基脯氨酸羟化，同时使α-酮戊二酸化学计量地脱羧生成琥珀酸和二氧化碳。已发现该酶的高度纯化制剂在没有肽基脯氨酸底物的情况下能使α-酮戊二酸脱羧。这种解偶联的脱羧反应速率仅为整个反应速率的一小部分，并且为了进行研究需要使用底物量的纯酶，以及氧气、亚铁离子和抗坏血酸。在这些条件下不会形成羟脯氨酸。已发现固定化的脯氨酰羟化酶抗血清能从酶制剂中去除这两种活性。然而，在孵育过程中添加游离抗血清仅抑制完整反应。聚（L-脯氨酸），一种脯氨酰羟化的特异性抑制剂，能增强α-酮戊二酸的解偶联脱羧反应，而其本身不会被羟化。所有这些发现证明，在没有肽基脯氨酸的情况下，α-酮戊二酸可以作为底物，并且很可能是氧气攻击的初始位点。在偶联反应中，酮酸的氧化形式，可能是过氧酸，然后攻击未羟化底物中的脯氨酰残基。
