Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen

The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.     

Photoaffinity labelling of the 2-oxoglutarate binding site of prolyl 4-hydroxylase with 5-azidopyridine-2-carboxylic acid

The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.     

Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii.

Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.     

The function of ascorbate with respect to prolyl 4-hydroxylase activity

1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.     

Hydroxylation of (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 by prolyl hydroxylase. Evidence for an asymmetric active site in the enzyme.

Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite.     

Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline

Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

A new prolyl hydroxylase acting on poly-L-proline, from suspension cultured cells of Vinca rosea

A new prolyl hydroxylase having a novel substrate specificity was isolated from the suspension-cultured cells of Vinca rosea. This enzyme was solubilized with 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1% Triton X-100, 0.3 M NaCl and 0.5 mM beta-mercaptoethanol from the membrane fractions of the cells, and was partially purified by (NH4)2SO4 fractionation and DEAE-Sephadex A-50 column chromatography. The enzyme preparation was found to require O2, Fe2+, ascorbate, alpha-ketoglutarate and poly-L-proline to attain maximum activity. The plant enzyme does not hydroxylate free proline and di-, tri- and tetra-L-proline, but hydroxylates octa-L-proline and poly-L-proline (Mr greater than 2000). Model peptides of unhydroxylated collagen, (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 are poor substrates for the plant enzyme. This means that the plant enzyme has a novel substrate specificity in regard to peptidyl substrate, and this differs from vertebrate prolyl hydroxylase, proline,2-oxoglutarate dioxygenase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase, EC 1.14.11.2).     

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin

In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.     

Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.     

Synthesis of 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D binding protein

Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.     

Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization

The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.     

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.     

Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.     

Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex.

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.