Prolyl hydroxylation

Kivirikko KI, Myllyla R, Pihlajaniemi T (1989), Protein hydroxylation prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 3 1609-1617. 

Ascorbic acid has been shown to increase collagen synthesis by fibroblasts in vitro (99, 280) and to maintain collagen synthesis In nonmitotic fibroblEists for extended periods (99). Prolyl hydroxylase, the enzyme hydroxylating prolyl and lysyl residues of procollagen, requires ascorbate to function in vitro (181), and the addition of ascorbic acid to tissue cultures stimulates the prolyl hydroxylase activity of fibroblasts (174). 

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

Very few post-translational modifications have been found on tropoelastin. However, hydroxylation of 25% of the proline residues is observed [10]. The enzymatic modification of proline to hydroxyproline (Hyp) is performed by prolyl hydroxylase [11]. The purpose of this hydroxylation remains unclear and it is even proposed that Hyps in tropoelastin are a by-product of collagen hydroxylation as this occurs in the same cellular compartment [8]. 

Hydroxylation of prolyl residues and some lysyl residues glycosylation of some hydroxylysyl residues... 

Jaakkoia, P. et al. Targeting of HlFalpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001, 292, 468-72. 

Epstein, A. C. et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001, 307, 43-54. 

Another important 2-OG dependent oxygenase in mammals is prolyl-4 hydroxylase, which catalyzes the hydroxylation of the proline residue in collagen (Scheme 5). This reaction is essential for the structure of the collagen triple helices (9,34 6). An overproduction of collagen is related to fibrotic diseases such as rheumatic arthritis. Thus collagen prolyl-4 hydroxylase is a target for therapeutics (34,36). 

Prolyl 4-hydroxylation is the most abundant posttranslational modification of collagens. 4-Hydroxylation of proline residues increases the stability of the triple helix and is a key element in the folding of the collagen triple helix. " In vertebrates, almost all the Yaa position prolines of the Gly-Xaa-Yaa repeat are modified to 4(I( )-hydroxylproline by the enzyme P4H (EC 1.14.11.2), a member of Fe(II)- and 2-oxoglutarate-dependent dioxygenases. This enzyme is an 0 2/ b2-type heterotetramer in which the / subunit is PDI (EC 5.3.4.1), which is a ubiquitous disulfide bond catalyst. The P4H a subunit needs the 13 subunit for solubility however, the 13 subunit, PDI, is soluble by itself and is present in excess in the ER. Three isoforms of the a subunit have been identified and shown to combine with PDI to form [a(I)]2/ 2) [< (II)]2/32> or [a(III)]2/32 tetramers, called the type... 

Oxoglutarate-Ee(ll) Oxy superfamily hydroxylation catalytic domain Prolyl 4-hydroxylase alpha subunit N-terminal domain superfamily... 

Selected prolines and lysines are hydroxylated by prolyl and lysyl hydroxylases. These enzymes, located in the RER, require ascorbate (vitamin C), deficiency of which produces scurvy. 

Answer E. The patient has many signs of scurvy from a vitamin C deficiency. The diet, which contains no fruits or vegetables, provides little vitamin C, Prolyl hydroxylase requires vitamin C, and in the absence of hydroxylation, the collagen a-chains do not form stable, mature collagen. The anemia may be due to poor iron absorption in the absence of ascorbate. 

Tschank, G., Brocks, D.G., Engelbart, K., Mohr, J., Baader, E., Gunzler, V. and Hanauskeabel, H.M. (1991) Inhibition of Prolyl Hydroxylation and Procollagen Processing in Chick-Embryo Calvaria by A Derivative of Pyridine-2,4-Dicarboxylate - Characterization of... 

It is reported that GPA-1734 inhibits prolyl and lysyl hydroxylations by Fe chelation and that the formation of non-dialyzable labeled hydroxyproline was inhibited 70% at a concentration of 50 pM, yet incorporation of proline into total liver protein was unaffected at this concentration (78BBA(538)328). GPA-1734 is a potent inhibitor of basement membrane synthesis (78MI21002). 

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. 

Hydroxylation of prolyl residues of pro-a-chains of collagen by prolyl hydroxylase. 

The active form of vitamin C is ascorbate acid (Figure 28.8). The main function of ascorbate is as a reducing agent in several different reac lions. Vitamin C has a well-documented role as a coenzyme in hydroxy lation reactions, for example, hydroxylation of prolyl- and lysyl-residues of collagen (see p. 47). Vitamin C is, therefore, required for the mainte nance of normal connective tissue, as well as for wound healing. Vitamin C also facilitates the absorption of dietary jron from the intestine. 

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