Oxoglutarate Linked Iron Containing Hydroxylases

A number of iron-containing hydroxylases (Table 1) share a common reaction mechanism, in which hydroxylation of the substrate is linked to decarbox-ylation of 2-oxoglutarate. Ascorbate is required for the activity of all of these enzymes, but it does not function as either a stoichiometric substrate or a conventional coenzyme (which would not be consumed in the reaction).

Proline and lysine hydroxylases are required for the postsynthetic modification of collagen, and proline hydroxylase also for the postsynthetic modification of osteocalcin in bone and the Clq component of complement. Aspartate ^-hydroxylase is required for the postsynthetic modification of protein C, the vitamin K-dependent protease which hydrolyzes activated factor V in the blood-clotting cascade. Trimethyllysine and 7-butyrobetaine hydroxylases are required for the synthesis of carnitine.

The best studied of this class of enzymes is procollagen proline hydroxylase; it is assumed that the others follow essentially the same mechanism. As shown in Figure 2, the first step is binding of oxygen to the enzyme-bound iron, followed by attack on the 2-oxoglutarate substrate, resulting in decarboxyla-tion to succinate, leaving a ferryl radical at the active site of the enzyme. This catalyzes the hydro-xylation 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 after about 5-10 s (about 15-30 cycles of enzyme action) the rate of reaction falls. The loss of activity is due 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

Table 1 Vitamin C-dependent, 2-oxoglutarate-linked hydroxylases

Aspartate ^-hydroxylase

EC 1

.14.11.16

7-Butyrobetaine hydroxylase

EC 1

.14.11.1

p-Hydroxyphenylpyruvate hydroxylase

EC 1

.14.11.27

Procollagen lysine hydroxylase

EC 1

.14.11.4

Procollagen proline 3-hydroxylase

EC 1

.14.11.7

Procollagen proline 4-hydroxylase

EC 1

.14.11.2

Pyrimidine deoxynucleotide dioxygenase

EC 1

.14.11.3

Thymidine dioxygenase

EC 1

.14.11.10

Thymine dioxygenase

EC 1

.14.11.6

Trimethyllysine hydroxylase

EC 1

COO-I

COO-

2-oxoglutarate

COO-

COO-

COO-

succinate

Enz uncoupled decarboxylation

peptide-incorporated hydroxyproline

Figure 2 The reaction of procollagen proline hydroxylase.

peptide-incorporated proline

2-oxoglutarate. Activity is only restored by ascor-bate, which reduces the iron back to Fe2+.

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