Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism.
From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors*; D011714:Pyrans / Q000494:pharmacology*; D011753:Pyrones / Q000494:pharmacology*
D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D011714:Pyrans; D011753:Pyrones; D011393:Procollagen-Proline Dioxygenase; C052808:coumalic acid