No abstract

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.

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.

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...

Lysyl hydroxylase (EC 1.14.11.4) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.Collagens are extracellular proteins found essentially in all tissues. They play a crucial role in maintenance of the structural integrity of tissues and in regulation of cellular behavior. The collagens, like other extracellular proteins, bind to growth factors and other regulatory components of cells and modulate cellular metabolism. To date, 19 genetically distinct collagen types have been identified (1-3). The collagen molecule is composed of three polypeptide chains, which coil around each other into a triple helical structure. Some of the collagen types, such as type I, II, and III collagens, have a rod-like structure without any interruptions in the helical region, whereas in the other types the triple-helical regions are interrupted with multiple short nonhelical sequences. Collagen molecules are aggregated in tissues into supramolecular structures such as fibrils, beaded filaments, or net-like or other kinds of structures depending on the type of collagen (3).The biosynthesis of collagen involves several post-translational modifications, which include hydroxylation of lysyl residues, galactosylation of hydroxylysyl residues, and glucosylation of galactosylhydroxylysyl residues. These reactions occur in the endoplasmic reticulum before triple helix formation. Hydroxylysine occurs in the Y position of the repeating XaaYaa-Gly triplet within the helical region of collagen molecules and also in the sequence of the nonhelical telopeptide regions of some collagen types when glycine is replaced either by serine or alanine (2, 4). The hydroxy groups of hydroxylysyl residues provide attachment sites for glycosyl residues, either the monosaccharide galactose or the disaccharide glucosylgalactose.The hydroxy groups also play a crucial role in the formation of inter-and intramolecular collagen cross-links. The biological role of the hydroxylysyl-linked carbohydrates, which are collagen-specific structures, is not clear. These carbohydrates point outward from the collagen helix and, thus, are located at the surface of the protein where they probably play an important role in lateral interactions between collagen triple helices and between collagen molecules and other extracellular ...