Prolines 3-hydroxylation reactions

Vitamin C is essential for the formation of collagen, the principal structural protein in skin, bone, tendons, and ligaments, being a cofactor in the hydroxylation of the amino acids proline to 4-hydroxyproline, and of lysine to 5-hydroxylysine. These hydroxyamino acids account for up to 25% of the collagen structure. Vitamin C is also associated with some other hydroxylation reactions, e.g. the hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) in the pathway to catecholamines (see Box 15.3). Deficiency leads to scurvy, a condition characterized by muscular pain, skin lesions, fragile blood vessels, bleeding gums, and tooth loss. Vitamin C also has valuable antioxidant properties (see Box 9.2), and these are exploited commercially in the food industries. 

The hydroxylation of specific Pro residues in procollagen, the precursor of collagen, requires the action of the enzyme prolyl 4-hydroxylase. This enzyme (Mt 240,000) is an a2/32 tetramer in all vertebrate sources. The proline-hydroxylating activity is found in the a subunits. (Researchers were surprised to find that the )3 subunits are identical to the enzyme protein disulfide isomerase (PDI p. 152) these subunits do not participate in the prolyl hydroxylation activity.) Each a subunit contains one atom of nonheme iron (Fe2+), and the enzyme is one of a class of hydroxylases that require a-ketoglutarate in their reactions. 

In the normal prolyl 4-hydroxylase reaction (Fig. 4a), one molecule of a-ketoglutarate and one of 02 bind to the enzyme. The a-ketoglutarate is oxidatively decarboxylated to form C02 and succinate. The remaining oxygen atom is then used to hydroxylate an appropriate Pro residue in procollagen. No ascorbate is needed in this reaction. However, prolyl 4-hydroxylase also catalyzes an oxidative decarboxylation of a-ketoglutarate that is not coupled to proline hydroxylation—and this is the reaction that requires ascorbate (Fig. 4b). During this reaction, the heme Fe2+ becomes oxidized, and the oxidized form of the enzyme is inactive—unable to hydroxylate proline. The ascorbate consumed in the reaction presumably functions to reduce the heme iron and restore enzyme activity. 

FIGURE 4 The reactions catalyzed by prolyl 4-hydroxylase, (a) The normal reaction, coupled to proline hydroxylation, which does not require ascorbate. The fate of the two oxygen atoms from 02 is shown in red. (b) The uncoupled reaction, in which a-ketoglutarate is oxidatively decarboxylated without hydroxylation of proline. Ascorbate is consumed stoichiometrically in this process as it is converted to dehydroascorbate. 

Cofactor for hydroxylation reactions, for example In procollagen Proline -> hydroxyproline Lysine -> hydroxylysine —Ot.QrT Y i —. ft... 

The fundamental role of ascorbic acid in metabolic processes is not well understood. There is some evidence that it may be involved in metabolic hydroxylation reactions of tyrosine, proline, and some steroid hormones, and in the cleavage-oxidation of homogentisic acid. Its function in these metabolic processes appears to be related to the ability of vitamin C to act as a reducing agent. 

Ascorbic acid (vitamin C fig. 10.16) is the reducing agent required to maintain the activity of a number of enzymes, most notably proline hydroxylase, which forms 4-hydroxyproline residues in collagen. Hydroxyproline (see fig. 10.16c) is not synthesized biologically as a free amino acid but rather is created by modification of proline residues already incorporated into collagen. The hydroxylation reaction occurs as the protein is synthesized in the endoplasmic reticulum. At least a third of the numerous proline residues in collagen are modified in this way, substantially increasing the resistance of the protein to thermal denaturation. 

The present work was undertaken in an attempt to shed light on the mechanism of hydroxyproline formation in collagen biosynthesis and on the site of action of ascorbate. It was recognized that these phenomena were very likely to be closely interrelated. Proline labeled with tritium was used because it was anticipated that tritium released from proline during hydroxylation would appear in the tissue water as tritiated water. The use of tritiated proline in such a system might then provide a new parameter for following the hydroxylation reaction, provided that the formation of tritiated water was stoichiometrically related to the formation of hydroxyproline. Such an approach might conceivably permit the demonstration of hydroxylation and peptide bond formation as separate chemical steps. 

The striking increases in the formation of tritiated water and tritiated hydroxyproline on in vitro addition of ascorbate are consistent with a function of this vitamin in hydroxylation—probably at step 3. The present results do not support a systemic ascorbic acid-mediated effect, the belief that ascorbic acid functions in the maintenance of collagen, or acts by stimulating maturation of the fibroblasts in the system under study here. The present data do not support the possibility that intermediates containing hydroxyproline accumulate in scurvy. The proposal that ascorbic acid is involved in the hydroxylation reaction itself is consistent with studies on the nonenzymatic hydroxylation of proline (4) and on enzymatic hydroxylation of other compounds (5, 20, 21, 44, 51). 

Ascorbic acid is a cofactor in various hydroxylation reactions. These reactions include the hydroxylation of proline residues in a variety of proteins, such as the... 

Another characteristic of ascorbic acid, CgHgOg, is that it is closely related chemically to glucose, C6Hi20g (Chaney, in Devlin, 1986, p. 1225). Whether or not this chemical resemblance has a desirable effect on some of its properties is fuel for speculation. For the record, its main biochemical role is said to be that of a reducing agent in certain important hydroxylation reactions, for example, of lysine and proline in protocollagen. It is therefore important in maintaining normal connective tissue. 

The hydroxylation reactions in collagen involve vitamin C. A symptom of extreme vitamin C deficiency, called scurvy, is the weakening of collagen fibers caused by the failure to hydroxylate proline and lysine. Consequences are as might be expected Lesions develop in skin and gums, and blood vessels weaken. The condition quickly improves with administration of vitamin C. 

Procollagen(I) is an example of a protein that undergoes extensive posttransla-tional modifications. Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues. These reactions occur after the protein has been synthesized (Fig. 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes, for example, prolyl hydroxylases and lysyl hydroxylase. Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose). 

Floyd, R.A. Zs-Nagy (1984). Formation of long lived hydroxyl free radical adducts of proline and hydroxyproline in a Fenton reaction. Biochimica Bio-physica Acta, 790, 94-7. 

Peptidyl hydroxyprohne and hydroxylysine are formed by hydroxylation of peptidyl proline or lysine in reactions catalyzed by mixed-function oxidases that require vitamin C as cofactor. The nutritional disease scurvy reflects impaired hydroxylation due to a deficiency of vitamin C. 

A number of iron-containing, ascorbate-requiring hydroxylases share a common reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of a-ketoglutarate (Figure 28-11). Many of these enzymes are involved in the modification of precursor proteins. Proline and lysine hydroxylases are required for the postsynthetic modification of procollagen to collagen, and prohne hydroxylase is also required in formation of osteocalcin and the Clq component of complement. Aspartate P-hydroxylase is required for the postsynthetic modification of the precursor of protein C, the vitamin K-dependent protease which hydrolyzes activated factor V in the blood clotting cascade. TrimethyUysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine. 

Figure 1.18 Reaction of proline, arginine, and lysine residues with hydroxyl radical results in oxidation of side-chain structures that form carbonyls. Both arginine and proline oxidation result in the same product.

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