Procollagen proline hydroxylase

Proline is incorporated into procollagen, the polypeptide precursor of collagen. In procollagen, proline is converted to hydroxyproline by the enzyme procollagen proline hydroxylase (Figure 21.4). In... 

The best studied of this class of enzymes is procollagen proline hydroxylase it is assumed that the... 

Procollagen proline 4-dioxygenase [Fe, ascorbate]— proline hydroxylase ... 

Procollagen proline 4-hydroxylase is the best studied of this class of enzymes it is assumed that the others have essentially the same mechanism, although proline and lysine hydroxylases show very little sequence homology (Kivirikko and Pihlajaniemi, 1998). Although 3-hydroxyproline is found only in collagen, 4-hydroxyproline and hydroxylysine are found in a variety of other proteins, including the Clq component of complement, osteocalcin, macrophage receptor proteins, and a variety of transmembrane and intercellular proteins and proteins of the cytoskeleton, as weU as some enzymes. 4-Hydroxyproline, but not hydroxylysine, also occurs in elastin. 

Proline hydroxylase has been isolated and characterized and is known to contain a mononuclear nonheme ferrous iron center that is the catalytic active site of the enzyme, coordinated by two histidine and one aspartate side chains. The requirement for Fe(II) is reflected in the in vivo sensitivity of collagen formation to chelators specific for ferrous ion (e g. 2,2 -dipyridyl). In addition to the catalytic metal cofactor, the reaction requires a reducing cosubstrate, 2-oxoglutarate, dioxygen, and the procollagen peptide (equation 1). 

Proline and lysine hydroxylases are required for the post-synthetic modification of procollagen in the formation of mature, insoluble, collagen, and proline hydroxylase is also required for the post-synthetic modification of the precursor proteins of osteocalcin and the Clq component of complement. 

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. 

The formation of Hyp and Hyl residues in procollagen is catalyzed by iron-containing oxygenases ( proline and lysine hydroxylase, EC 1.14.11.1/2). Ascorbate is required to maintain their function. Most of the symptoms of the vitamin C deficiency disease scurvy (see p. 368) are explained by disturbed collagen biosynthesis. 

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. 

Before this "maturation" can occur there must be other modifications to procollagen. These begin while the peptide chains are still attached to ribosomes of the rough ER. Hydroxylases (Chapter 18) localized in the membranous vesicles of the ER convert some of the proline and lysine residues of the procollagen chains into 4-hydroxyproIine625 629 630 and hydroxylysine (Eqs. 8-6 and 8-7). Lesser amounts of 3-hydroxypro-line are formed. About 100 molecules of 4-hydrox-yproline and 50 of 5-hydroxylysine are created in each al chain. 

The first evidence for PDI having a different role came from Pihla-janiemi etal. (1987) during their study of the enzyme prolyl 4-hydroxylase (P4H). This enzyme is localized in the ER and catalyzes the hydroxylation of proline residues in nascent procollagen polypeptides. The enzyme is a tetramer composed of two a and two jS subunits (Kivirikko et al., 1989). Antibodies raised against the holoenzyme were used to screen a human gtll expression library and clones were isolated that coded for the /3 subunit. After sequencing it was revealed that the human /3 subunit was 94% identical with the rat PDI sequence. Southern blot analysis identified... 

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