Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.11.2 (prolyl hydroxylase)
 1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.
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PMID:The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity. 255 1

The enzymatically catalyzed formation of 4-hydroxyproline plays a key role in the intracellular biosynthesis of collagen, since a critical number of 4-hydroxyprolyl residues is required for synthesis and secretion of triple-helical procollagen molecules under physiologic conditions. The enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, prolyl 4-hydroxylase, requires ferrous ion, alpha-ketoglutarate, and ascorbate for its activity. 3,4-Dihydroxybenzoic acid has been known to act as potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme. 3,4-Dihydroxybenzoic acid, however, is a poor inhibitor of prolyl hydroxylation in intact cells, probably due to its polarity not allowing it to enter the cells. In this study, several hydrophobic modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately 0.4 mM. Ethyl 3,4-dihydroxybenzoate had little, if any, effect on the hydroxylation of lysyl residues, and it did not affect total protein synthesis or DNA replication in these cells. To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, its efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was also tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel means of reducing collagen deposition in tissues in fibrotic diseases, such as scleroderma.
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PMID:Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures by ethyl 3,4-dihydroxybenzoate. 282 18

Two enzyme activities concerned with collagen disaccharide unit metabolism (UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase) have been studied in the thoracic aortic wall together with 4-prolyl hydroxylase activity and 4-hydroxyproline content in spontaneously hypertensive rats (SHR) at the prehypertensive, hypertensive and sustained hypertensive stages (respectively 32 days, 12 weeks and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. Negative regressions of all three specific activities as function of age were observed. The most striking difference observed between the SHR and the WKY was the increase of glucosyltransferase specific activity, already found at the prehypertensive stage and continuing thereafter; the glucohydrolase specific activity was increased only during the establishment of hypertension whereas no modification was found with prolyl hydroxylase at any stage. However, a diminution of hydroxyproline concentration was seen at all ages while total hydroxyproline mass remained unaffected. The alterations of the aortic collagen metabolism observed in the ACR recall those seen in the SHR at the prehypertensive stage: the only significant modification was that of glucosyltransferase activity. Correlation was found between glucosyltransferase activity and blood pressure level in the two animal models.
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PMID:Aorta collagen metabolism in spontaneously hypertensive and aortic-constricted rats: variations in enzyme activities concerned with disaccharide unit synthesis and degradation according to blood pressure and age. 293 74

Collagen biosynthesis involves many unique post-translational events. Inhibition of some of these will lead either to decreased formation of the extracellular collagen fibres or to an accumulation of fibres with altered functional properties. The events that would seem most suitable targets for chemical regulation are triple helix formation, the cleavage of propeptides from the procollagen molecules and cross-link formation. Attempts have recently been made to develop inhibitors of prolyl 4-hydroxylase in particular, as inhibition of this enzyme will prevent triple helix formation and thus lead to a non-functional protein. Prolyl 4-hydroxylase is inhibited competitively with respect to ferrous ion by several bivalent cations, especially zinc, with respect to 2-oxoglutarate by pyridine 2,5-dicarboxylate, pyridine 2,4-dicarboxylate, 3,4-dihydroxybenzoate and many related compounds, with respect to oxygen by superoxide dismutase-active copper chelates and with respect to the peptide substrate by a number of peptides. Triple helix formation can also be inhibited by administering certain proline analogues such as cis-4-hydroxyproline and L-azetidine-2-carboxylic acid, which are incorporated into proteins in place of proline. Only preliminary data are available on the possibilities for using any of these substances to inhibit collagen accumulation in fibrotic processes.
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PMID:Synthesis of collagen: chemical regulation of post-translational events. 299 12

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.
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PMID:Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene. 303 2

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.
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PMID:Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action. 303 58

3-prolyl hydroxylase activity measurements have already been described by Kivirikko and al, using specific methods. The aim of the present work was to show that the specific and rapid method used for 4-prolyl hydroxylase activity measurement, involving protocollagen [3H-4] proline (measuring of tritiated water enzymatically obtained), could be used for 3-prolyl hydroxylase activity estimation on the same sample: tritiated water enzymatically produced by 4-prolyl hydroxylase was collected by distillation, and the amino acids enzymatically modified were analysed after HCl 6 N hydrolysis of dried incubation medium, by cation exchange chromatography. The characterization of enzymatically obtained 3-hydroxyproline was performed using three means. The elution peaks reported were in the same position as the elution peak of pure 3-hydroxyproline and 4-hydroxyproline. Moreover, tritiated 3-hydroxyproline and 4-hydroxyproline were obtained only after incubation of labelled substrate with crude preparation of prolyl hydroxylases from chick embryos. Some possible artefacts such as dicetopiperazines and pyrrol-2-carboxylic acid have been shown to be distinguished chromatographically from 3-hydroxyproline and 4-hydroxyproline. The high ratio of measured (Formula: see text) activities, near 5.5 p. cent, is discussed.
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PMID:[Simultaneous characterizations of 3-prolylhydroxylase and 4-prolylhydroxylase activities by ion exchange chromatography]. 624 67

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), and the concentration of 4-hydroxyproline were measured in red and white parts of quadriceps femoris muscle of mice after 3, 10, and 20 sessions of daily endurance training. The activities of PH and GGT increased in the red part of the muscle after training for 3 and 10 times and returned to the control level after 20 training sessions. In the white muscle the increase of PH activity was less than in the red muscle. No alteration in GGT activity was observed in the white muscle. The concentration of hydroxyproline was unchanged in the both types of skeletal muscle. The results suggest that collagen turnover in leg muscles may be enhanced during the early phase of adaptation to endurance training. The enhancement is more prominent in red than in white skeletal muscle.
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PMID:Increased activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, enzymes of collagen biosynthesis, in skeletal muscle of endurance-trained mice. 632 85

Concomitant hydroxylation of proline and lysine residues in protocollagen was studied using purified enzymes. The data suggest that prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2-oxoglutarate; oxygen 5-oxidoreductase, EC 1.14.11.4) are competing for the protocollagen substrate, this competition resulting in an inhibition of the lysyl hydroxylase but not of the prolyl 4-hydroxylase reaction. When the same protocollagen was used for these hydroxylases, the affinity of prolyl 4-hydroxylase to the protocollagen substrate was about 2-fold higher than that of lysyl hydroxylase. Hydroxylation of lysine residues in protocollagen had no effect on the affinity of prolyl 4-hydroxylase, whereas hydroxylation of proline residues decreased the affinity of lysyl hydroxylase to one-half of the value determined before the hydroxylation. When enzyme preparations containing different ratios of lysyl hydroxylase activity to prolyl 4-hydroxylase activity were used to hydroxylase protocollagen substrate, it was found that in the case of a low ratio the hydroxylation of lysine residues seemed to proceed only after a short lag period. Accordingly, it seems probable that most proline residues are hydroxylated to 4-hydroxyproline residues before hydroxylation of lysine residues if the prolyl 4-hydroxylase and lysyl hydroxylase are present as free enzymes competing for the same protocollagen substrate.
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PMID:Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro. 633 20

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer in which the alpha subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human alpha subunit individually to serine and expressed the mutant alpha subunits together with the wild-type beta subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Fe2+ to a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Fe2+ binding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.
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PMID:Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity. 773 Mar 75


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