Mechanism of the Prolyl Hydroxylase Reaction

Abstract

The co‐substrate requirements of prolyl hydroxylase were studied with pure enzyme from chick embryos. No hydroxylation occurred without added Fe2+, indicating that the enzyme does not retain iron sufficiently to catalyze any reaction. Zn2+ was an effective competitive inhibitor with respect to Fe2+, but was noncompetitive with respect to the polypeptide substrate and 2‐oxoglutarate, suggesting that it replaced iron in the active site of the enzyme.The enzyme catalyzed the uncoupled decarboxylation of 2‐oxoglutarate at a rate of about 4 mol CO2 formed (mol enzyme)−1 min−1 in the presence of Fe2+, O2, and ascorbate but in the absence of the polypeptide substrate. This rate was about 1/80 of that observed in the presence of the substrate. Several compounds inhibited the enzyme competitively with respect to 2‐oxoglutarate but non‐competitively with respect to Fe2+. It seems that these two co‐substrates become bound at separate sites on the enzyme, and additional data suggested that these are distinct from the binding site of the polypeptide substrate.The reaction was completely dependent on O2. Nitroblue tetrazolium was a competitive inhibitor with respect to O2, but noncompetitive with respect to the polypeptide substrate and all other co‐substrates. Epinehrine also inhibited the enzyme, but this inhibition was competitive with respect to Fe2+. The results suggest that nitroblue tetrazolium consumed an activated form of oxygen, whereas epinephrine acted primarily by binding Fe2+.The reaction was completely dependent on ascorbate, and in contrast to previous data, this could not be significantly replaced by tetrahydrofolic acid or dithiothreitol. Dehydroascorbate replaced ascorbate in the presence of dithiothreitol but not in its absence. The results also indicate that ascorbate is not stoichiometrically consumed during the reaction.