The influence of 2-oxoglutarate on the activity of prolyl 4-hydroxylase

Abstract

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 Fe 2+ for 10s causes partial inactivation of the enzyme which is not reversed by subsequent addition of Fe 2+. 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 15s with Fe 2+ (5μM). 3. Under turnover conditions prolyl 4-hydroxylase does not release iron. 4. The inactivation brought about by pre-incubation with 2-oxoglutarate and 0 2 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 Fe 2+. 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 15s 2-oxoglutarate and finally (Pro-Pro-Gly) 5 to start the reaction.