Prolyl hydroxylase reaction

Figure 28-11. The prolyl hydroxylase reaction. The substrate is a proline-rich peptide. During the course of the reaction, molecular oxygen is incorporated into both succinate and proline. Lysyl hydroxylase catalyzes an analogous reaction.

The alternate mechanism proposed by Hamilton59) seems more attractive since it is equivalent to the mechanism proposed for the prolyl hydroxylase reaction discussed earlier. 

Reduction of the asparagine s hydroxylase reaction allows the HIFa to do its job, and reduction of the prolyl hydroxylase reaction stops degradation of the HIFa by the ubiquitin-linked pathway. Many researchers believe that these two reactions are the mechanism for oxygen sensing, but others believe they are just incidental to the true mechanism, which has yet to be discovered. 

Myllila, R., Kuutti-Savoilanen, E. R., and Kivirikko, K. L, 1978, The role of ascorbate in the prolyl-hydroxylase reaction, Biochem. Biophys. Res. Commun. 83 441-448. 

Aseorbate has an important role in the proeollagen prolyl hydroxylase reaction, most probably allowing recovery of the enzyme following abortive... 

FIGURE 6.17 Hydroxylation of proUne residnes is catalyzed by prolyl hydroxylase. The reaction requires -ketoglntarate and ascorbic acid (vitamin C). 

Mineral dust-induced ROMs contributes to pulmonary fibrosis, malignancy, hypersensitivity and emphysema (Doelman etctl., 1990 Kamp etui., 1992). The involvement of ROMs in pulmonary fibrotic reactions is indicated by the participation of PMN oxidants in the autoactivation of latent coUagenase (Weiss et al., 1985). Prolyl hydroxylase, a key enzyme in collagen fibril formation, has been shown to be dependent on the reaction of superoxide with prolyl residues (Myllyla et al., 1979). 

Myllyla, R., Schubotz, L.M., Weser, U. and Kivirikko, K.I. (1979). Involvement of superoxide in the prolyl and lysyl hydroxylase reactions. Biochem. Biophy. Res. Commun. 89, 98-102. 

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 13.G. Reaction sequence of prolyl hydroxylase (EC 1.14.11.2). Enz, enzyme.

The enzyme that catalyzes this reaction, lysine hydroxylase, is, like prolyl hydroxylase, a Fe(II)/2-oxoglutarate dependent dioxygenase but exhibits a distinct substrate specificity. 

The hydroxylation reactions are catalyzed by three enzymes prolyl 4-hydroxylase (usually known as prolyl hydroxylase), prolyl 3-hydroxylase, and lysyl hydroxylase. These enzymes are located within the cistemae or rough endoplasmic reticulum as the procollagen chains enter this compartment, the hydroxylations begin. 

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). 

Poly(VPGVG) is a substrate for the natural enzyme prolyl hydroxylase, which uses molecular oxygen and the cofactor vitamin C (ascorbic acid) for the reaction. Figure 7.49 also contains the temperature profile for fiber formation that results from hydroxylation by prolyl hydroxylase. When 100% hydroxylated as in poly(Val-Hyp-Gly-Val-Gly), T, is about 65° C. An estimated 1 % hydroxylation by prolyl hydroxylase results in a value of T, of about 40°C. As shown in Figure 5.1C, T is just at the very onset of the aggregation, such that raising the value of T, from about 30° to about 40° C would prevent assembly into fibers at body temperature. Prolyl hydroxylation of the polypentapeptide model of elastin impairs fiber formation at physiological temperature. 

Counts, D. F., G. J. Cardinale, and S. Udenfriend Prolyl Hydroxylase Half Reaction Peptidyl Prolyl-independent Decarboxylation of a-Ketoglutarate. Proc. Natl Acad. Sci. U.S.A. 75, 2145 (1978). 

Although there seems to be no doubt that ascorbic acid facilitates collagen synthesis in vitro and in vivo, the exact role of vitamin C in the proline hydroxylase is still debated. When purified proline hydroxylase is used, reducing substances other than ascorbic acid can participate in the reaction (e.g., tetrahydrofolate or dithiothreitol). Inasmuch as an inactive prolyl hydroxylase has been found, it has been suggested, but not convincingly established, that ascorbic acid participates in the conversion of an inactive to an active form of the enzyme. 

One very important factor for the activity of 2-oxoglutarate enzymes is the availability of Fe. The addition of Fe to the reaction mixture is necessary for full activity, but the enzymic activity in the absence of added Fe varies considerably for the preparations reported in the literature (Table 3). This was found for prolyl hydroxylase also and attributed to the release of the metal from the active site to different degrees during purification [18]. Another reason for the Fe " requirement may be the presence of chelating agents, such as phosphate buffer, in the assay mixture. The effect of chelating agents will be discussed first. 

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