Inhibition of lysyl hydroxylase and prolyl 3-hydroxylase was studied with 23 selected aromatic and aliphatic structural analogues of 2-oxoglutarate and the results were compared with those previously reported for prolyl 4-hydroxylase. All the compounds inhibited the hydroxylases competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fe2+ and the peptide substrate. The inhibition patterns for the three collagen hydroxylases were basically similar, but certain differences in detail emerged. One systematic difference was that lysyl hydroxylase had a higher Ki for almost all the compounds than had the two prolyl hydroxylases. Another interesting difference was that pyridine-2,4-dicarboxylate was the most potent inhibitor of lysyl hydroxylase and prolyl 3-hydroxylase, with Ki values of 50 microM and 3 microM respectively, whereas pyridine-2,5-dicarboxylate was the most potent inhibitor of prolyl 4-hydroxylase. These and other data suggest that the three collagen hydroxylases have similar but not identical 2-oxoglutarate-binding sites. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate and their corresponding benzene derivatives were also found to inhibit 2-oxoglutarate dehydrogenase, but with this enzyme, unlike the collagen hydroxylases, no distinct difference in the Ki values was found between the corresponding pyridine and benzene derivatives. This demonstrates the importance of the metal ion for the binding of various compounds at the 2-oxoglutarate-binding site of the collagen hydroxylases. 2-Oxoadipate was shown to replace 2-oxoglutarate in the lysyl hydroxylase and 2-oxoglutarate dehydrogenase reactions, as has previously been reported for prolyl 4-hydroxylase, whereas no other 2-oxo acid tested had any co-substrate activity. The 2-oxoglutarate-binding site of these enzymes is thus flexible to a certain degree, as it can accommodate molecules of different shapes and volumes. On the basis of the present data pyridine-2,5-dicarboxylate seems to be a quite specific inhibitor of prolyl 4-hydroxylase, the Ki for 2-oxoglutarate dehydrogenase being about 4000-fold higher.
Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. LMimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases. (J. Clin. Invest. 1995. 95:446-455.)
A series of synthetic peptides was prepared and used to study protocollagen lysine hydroxylase, the enzyme which synthesizes the hydroxylysine in collagen. The enzyme was purified 150-to 250-fold from chick embryo extracts and it was completely free of protocollagen proline hydroxylase activity. Several synthetic peptides competitively prevented the hydroxylation of lysine-labeled [ 14C]protocollagen by protocollagen lysine hydroxylase. When the peptides were examined directly as substrates for the enzyme, the synthesis of measurable amounts of hydroxylysine was observed for the first time. The synthesis of hydroxylysine was accompanied by the conversion of a-ketoglutarate to C02 and succinate, indicating that the enzyme belongs to the new class of oxygenases which decarboxylate a-ketoglutarate during substrate hydroxylation. The release of [14C]COr from [l-14Cl]a-ketoglutarate was equimolar with the synthesis of hydroxylysine and therefore the amount of ['CC]CO2 released could be used as a simple assay for the enzymic reaction. Three peptides were synthesized which had amino acid sequences comparable to amino acid sequences around glycosylated hydroxylysine in collagen. The peptide "L-I" had the sequence Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-GlyPhe-Ser-Gly, "L-11" the sequence Ala-Arg-Gly-Met-Lys-GlyHis-Arg-Gly-(Pro-Pro-Gly),, and "L-111" the sequence (ProPro-Gly),-Ala-Arg-Gly-Met-Lys-Gly-His-Arg -Gly -(Prn -Pro-
The chromosomal location of the human gene coding for both the β-subunit of prolyl 4-hydroxylase (P4HB) and the enzyme disulfide isomerase (PDI) was determined using mouse × human somatic cell hybrids and three different methods for identifying either the human P4HB/PDI protein or the respective gene: immunoblotting with species-specific monoclonal antibodies; radioimmunoassay with species-specific polyclonal antibodies; and (3) Southern blotting after cleavage of the DNA with EcoRI, HindIII, or BamHI, followed by hybridization with a mixture of two cDNA probes for human P4HB. All three methods gave identical data, demonstrating complete cosegregation of the human protein or its gene in all 17 cell hybrids tested with human chromosome 17. A cell hybrid lacking an intact chromosome 17 localized the gene to 17p11→qter.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.