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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 kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs of Fe2+, 2-oxoglutarate, 0, and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concentration was varied at different fixed 2-oxoglutarate concentrations in the presence of saturating 0, concentration. Poly(L-proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe2+ and 2-oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe2+ and 2-oxoglutarate. Succinate, CO, and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2-oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent K, and Ks values for the substrates and Ki values for the inhibitors are given.These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe2+, 2-oxoglutarate, 0, and the polypeptide substrate to the enzyme in this order, the binding of Fe2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly(L-proline) under certain conditions, forming dead-end complexes. The products are released only after the hydroxylation, possibly in the order : the hydroxylated polypeptide, CO, and succinate. Ascorbate may react either with enzyme.Fe before the release of Fe2+ or with free enzyme before the binding of Fe2+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2-oxoglutarate dioxygenases.Prolyl hydroxylase belongs to the group of 2-0x0-glutarate dioxygenases, other members of which include lysyl hydroxylase, 4-butyrobetaine hydroxylase, thymine 7-hydroxylase, pyrimidine deoxyribonucleoside 2'-hydroxylase and p-hydroxyphenylpyruvate hydroxylase (for reviews, see [l -41). These enzymes require molecular oxygen, ferrous iron, 2-oxoglutarate and a reducing agent. p-Hydroxyphenylpyruvate hydroxylase, the substrate of which contains an 0x0 group, differs from the other enzymes in that it does not need 2-oxoglutarate but has apparently a similar reaction mechanism. The 2-oxoglutarate is stoichiometrically decarboxylated during these reactions [ 1 -61, Abbreviation. (Pro-Pro-Gly), , a linear copolymer composed of regular alternating sequences of L-proline-L-proline-glycine.Enzyme. Prolyl hy...

An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(L-proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(L-proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration.The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82 %, and the purified preparations synthesized from 59.3 to 91.5 pmol hydroxyproline per mg enzyme per h at 37 "C with a saturating concentration of (Pro-Pro-Gly), as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.Prolyl hydroxylase catalyzes the synthesis of hydroxyproline .in collagen by hydroxylation of prolyl residues in peptide linkages (for recent reviews, see [2 -51). The reaction requires 2-oxoglutarate7 molecular oxygen, ferrous iron and a reducing agent such as ascorbate [2-71. The 2-oxoglutarate is stoichiometrically decarboxylated during the hydroxylation of prolyl residues [8,9]. The enzyme does not hydroxylate free proline [7] or proline in the tripeptide Gly-ProPro [7], but a single sequence of -X-Pro-Gly-fulfills a minimum requirement for recognition [lo-121. Studies with a series of peptides with the general structure (X-Pro-Gly), demonstrated the decisive influence of chain length on interaction with the enzyme, in that longer peptides had lower K , values A preliminary report of the results has been presented [I].Abbreviation. (Pro-Pro-Gly),, a linear copolymer composed of regular alternating sequences of L-proline-L-prolineglycine.Enzyme. Prolyl hydroxylase or prolyl-glycyl-peptide, 2-oxoglutarate oxidoreductase (Chydroxylating) (EC 1.14.11.2).than shorter peptides of the same structure [12-151. Underhydroxylated collagen from the cuticle of Ascaris lumbricoides also serves as a good substrate [8,16]. Poly(L-proline) is not a substrate, but is an effective competitive inhibitor [13,14,20]. The affinity of the enzyme for poly(L-proline) increases markedly with increasing polymer length up to a molecular weight of about 20000 [21].The enzyme was initially purified to near homogeneity from chick embryo extract [17,18] and newborn rat skin [ 191 by procedures involving fractionating precipitation, ion-exchange chromatography and gel filtration. Subsequently, the high affinity of prolyl hydroxylase for its polypeptide substrates [22] was utilized to develop an affinity column pro...