Oxoglutarate-Linked Iron-Containing Hydroxylases

As shown in Table 13.1, a number of iron-containing hydroxylases share an unusual reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of 2-oxoglutarate. Proline and lysine hydroxylases are required for the postsynthetic modification of collagen, and proline hydroxylase also for the postsynthetic modification of osteocalcin (Section 5.3.3) and other proteins. Aspartate /3-hydroxylase is required for the postsynthetic modification of protein C, the vitamin K-dependent protease that hydrolyzes activated Factor V in the blood clotting cascade (Section 5.3.2). Trimethyllysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine (Section 14.1.1). 

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. 

In collagen, hydroxyproline stabilizes the triple helix structure by forming hydrogen bonds via water between adjacent chains or regions of the same chain. Hydroxylysine provides sites for glycosylation of proteins, and is essential for stabilization of intermolecular cross-links formed by reaction between lysine or hydroxylysine aldehyde and the e-amino group of lysine or hydroxylysine. 

Proline and lysine hydroxylases are found in the lumen of rough endoplasmic reticulum. Hydroxylation of the peptide substrate occurs both cotransla-tionaUy and later as a postsynthetic modification. The enzymes act only on peptides and not on free amino acids. 

It has long been known that ascorbate is oxidized during the reaction, but not stoichiometricaUy with hydroxylation of proline and decarboxylation of 2-oxoglutarate. The purified enzyme is active in the absence of ascorbate but, after 5 to 10 seconds (about 15 to 30 cycles of enzyme action), the rate of reaction begins to fall. The loss of activity is from a side reaction of the highly reactive ferryl radical in which the iron is oxidized to Fe +, which is catalyticaUy inactive - so-called uncoupled decarboxylation of 2-oxoglutarate. Activity is only restored by ascorbate, which reduces the iron back to Fe + (Kivirikko and Pihlajaniemi, 1998). 

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Ascorbic acid has specific and weU-deflned roles in two classes of enzymes the copper-containing hydroxylases (such as dopamine /3-hydroxylase and peptidyl glycine hydroxylase) and the 2-oxoglutarate-linked iron-containing hydroxylases, of which the best studied are the proline and lysine hydroxylases involved in maturation of connective tissue (and other) proteins. 

A number of iron-containing hydroxylases (Table 1) share a common reaction mechanism, in which hydroxylation of the substrate is linked to decarboxylation of 2-oxoglutarate. Ascorbate is required for... 

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