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 Fez+, indicating that the enzyme does not retain iron sufficiently to catalyze any reaction. Zn2+ was an effective competitive inhibitor with respect to Fez+, 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 CO, formed (mol enzyme)-l min-' in the presence of Fez+, 0,, 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 noncompetitively with respect to Fez+. 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 0,. Nitroblue tetrazolium was a competitive inhibitor with respect to 0,, but noncompetitive with respect to the polypeptide substrate and all other cosubstrates. Epinephrine also inhibited the enzyme, but this inhibition was competitive with respect to Fez+. The results suggest that nitroblue tetrazolium consumed an activated form of oxygen, whereas epinephrine acted primarily by binding FezThe 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.Prolyl hydroxylase catalyzes the synthesis of hydroxyproline in collagen by the hydroxylation of certain prolyl residues in peptide linkages (for recent reviews, see [I -41). The enzyme has been isolated as a homogeneous protein by affinity chromatography from three sources [5 -81, and found to be a tetramer with a molecular weight of about 240000 [5 -91 and consisting of two different types of monomers with molecular weights of about 60000 and 64000 [5 -81. The enzyme does not hydroxylate free proline, and the minimum sequence requirement is an -X-Pro-Glytriplet [I -41. The hydroxylation of prolyl residues in this sequence is influenced by the nature of the amino acid in the X position of the triplet, the nature of the amino acids in the adjacent sequences, and the chain Abbreviation. (Pro-Pro-Gly),, a linear copolymer composed of regular alternating sequences of L-proline-L-proline-glycine.Enzyme. Prolyl hydroxylase or prolyl-glycyl-peptide,2-0~0-glutarate: oxygen oxidoreductase (4-hydroxylating) (EC 1.14.1 1.2). length and conformation of the peptide substrate [1,3,41. Prolyl hydroxylase requires 2-oxoglutarate, molecular oxygen, ferrous iron and a reducing agent [ 1 -4, 10 -151. The 2...

The 2‐oxoglutarate binding site of prolyl 4‐hydroxylase

“…This mode of 2-oxoglutarate chelation to subsite I1 produces a significant reduction in electron density at the spz hybridized C2 atom and constitutes its carbocationic character. thus increasing the susceptibility of C2 to nucleophilic attack by the noncoordinated oxygen atom pounds have previously been identified as prolyl4-hydroxylase inhibitors competitive with respect to 2-oxoglutarate [4]. The data reported here indicate that a number of aliphatic and aromatic compounds inhibit prolyl4-hydroxylase with respect to this cosubstrate, the results testifying to the existence of subsites I and 11, and an additional hydrophobic binding site 111.…”

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates

“…Succinate, oxaloacetate, and citrate have also been reported (26,27) to inhibit the C-P4Hs, their K i values for the C-P4H isoenzyme I being 400, 100, and 450 M, respectively (Table 1). Our current data indicate that fumarate is an additional C-P4H-I inhibitor, with a K i of 190 M ( Table 1).…”

Partial Purification and Characterization of the Gibberellin A20 3β-Hydroxylase from Seeds of Phaseolus vulgaris

“…Thus, uncharacteristically, the association between Fe2+ and the enzyme appeared to be weak. For almost all documented 2-oxoglutarate dependent dioxygenases, residual activity, ranging from 10 to 100%, was measurable in the absence of Fe2+ (28). In the extreme cases, Fe2+ was bound so tightly that chelators such as a,a-bipyridyl failed to abstract protein-bound Fe2' and thus inhibit enzyme activity (20).…”