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 f -hydroxylase is required for the postsynthetic modification of protein C, the vitamin K-dependent protease that hydrolyzes activated Factor Vin 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 C1q component of complement, osteocalcin, macrophage receptor proteins, and a variety of transmembrane and intercellular proteins and proteins of the cytoskeleton, as well 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 hydrox-ylysine.

Figure 13.6. Reaction sequence ofprolyl hydroxylase (EC Enz, enzyme.

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

As shown in Figure 13.6, the first step is binding of oxygen to the enzyme-bound iron, followed by attack on the 2-oxoglutarate substrate, resulting in decarboxylation to succinate, leaving a ferryl radical at the active site of the enzyme. This catalyzes the hydroxylation of proline, restoring the free iron to undergo further reaction with oxygen.

It has long been known that ascorbate is oxidized during the reaction, but not stoichiometrically with hydroxylation of proline and decarboxylation of 2-oxoglutarate. The purified enzyme is active in the absence of ascorbate; but, alter 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 Fe3+, which is catalytically inactive - so-called uncoupled decarboxylation of 2-oxoglutarate. Activity is only restored by ascorbate, which reduces the iron back to Fe2+ (Kivirikko and Pihlajaniemi, 1998).

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