Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism.
Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe2+, alpha-ketoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+, in which Fe2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO2 and succinate or after the release of all products, including Fe2+, and dehydroascorbate is released before the binding of Fe2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe2+, alpha-ketoglutarate, O2 and ascorbate respectively, and epinephrine non-competitively with regard to all cosubstrates. Apparent Ki values are given for the product and other inhibitors.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D002642:Chick Embryo; D003683:Dehydroascorbic Acid / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D006713:Homogentisic Acid / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D009580:Nitroblue Tetrazolium / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000037:antagonists & inhibitors*; D013386:Succinates / Q000494:pharmacology
D010455:Peptides; D013386:Succinates; D002245:Carbon Dioxide; D009580:Nitroblue Tetrazolium; D006899:Mixed Function Oxygenases; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D006713:Homogentisic Acid; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid; D004837:Epinephrine