The influence of 2-oxoglutarate on the activity of prolyl 4-hydroxylase.
1. Treatment of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with 2-oxoglutarate in the absence of added Fe2+ for 10 s causes partial inactivation of the enzyme which is not reversed by subsequent addition of Fe2+. It appears that 2-oxoglutarate prevents loss of enzyme-bound iron and prevents access of added iron to its binding site. 2. For optimal enzyme activity the enzyme should be preincubated for 15 s with Fe2+ (5 microM). 3. Under turnover conditions prolyl 4-hydroxylase does not release iron. 4. The inactivation brought about by pre-incubation with 2-oxoglutarate and O2 in the absence of ascorbate is partly reversed by removal of 2-oxoglutarate. 5. It is proposed that dead-end complex formation with 2-oxoglutarate is responsible for the inactivation of the enzyme by 2-oxoglutarate in the absence of either ascorbate or Fe2+. 6. Optimal enzyme activity is obtained if the reactants are added to the reaction medium in the following order: enzyme, iron and ascorbate in any order, then after at least 15 s 2-oxoglutarate and finally (Pro-Pro-Gly)5 to start the reaction.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002642:Chick Embryo; D005296:Ferrous Compounds / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D011393:Procollagen-Proline Dioxygenase; D001205:Ascorbic Acid