Ketoglutarate decarboxylating hydroxylase

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 a-ketoacid-dependent enzymes are distinguished from other non-haem iron enzymes by their absolute requirement for an a-ketoacid cofactor as well as Fe(II) and O2 for activity. They catalyse two types of reaction (Table 2.3), hydroxyla-tion and oxidation. In both, the a-ketoglutarate is decarboxylated and one oxygen atom introduced into the succinate formed in the hydroxylases, the other oxygen atom is introduced into the substrate, while in the oxidases it is found in water, together with the cyclized product. In general these enzymes require one equivalent of Fe(II) an a-ketoacid, usually a-ketoglutarate and ascorbate. Examples of these enzymes include proline 4-hydroxylase, prolyl and lysyl hydroxylase, which... 

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. 

Of all the enzymes of the carnitine biosynthetic pathway, yBBH is the best-studied enzyme. Like e-N-trimethyllysine hydroxylase, y-BBH is a non-heme ferrous-iron dioxygenase that requires a-ketoglutarate, Fe and molecular oxygen as cofactors. In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of a-ketoglutarate. Ascorbate is needed to maintain iron in die reduced state. yBBHhas been isolated from various sources including human kidney, " catf and rat liver and the bacterium Pseudomonas AKl. A common problem in the purification of this enzyme from mammalian tissues is the poor stability of the protein. We, therefore, determined the optimal storage conditions and subsequently pmified ybutyrobetaine hydroxylase from rat liver. 

Counts, D. F., G. J. Cardinale, and S. Udenfriend Prolyl Hydroxylase Half Reaction Peptidyl Prolyl-independent Decarboxylation of a-Ketoglutarate. Proc. Natl Acad. Sci. U.S.A. 75, 2145 (1978). 

As already mentioned, collagen contains the unique amino acids hy-droxyproline and hydroxylysine, which are necessary for the stability of the molecule and for its complete maturation. The synthesis of these amino acids occurs posttranslationally during the assembly of the polypeptidic chain (Uitto and Proc-kop, 1974), is independent of the age (Brinckmann et al., 1994), and is catalyzed by prolyl and lysyl hydroxylases in the presence of oxygen, a-ketoglutarate, ferrous ions, and ascorbic acid (Hutton et al., 1967 Kivirikko and Prockop, 1967). Ascorbic acid has been found to be specifically required for the decarboxylation of a-ketoglutarate in the proly 1-4-hydroxylase reaction, where it may act as a com-... 

Recently George Gardinale has been able to show that prolyl hydroxylase can catalyze the oxidative decarboxylation of a-ketoglutarate in the absence of a prolyl substrate,>at a lower but measurable rate. The same cofactors, Fe and ascorbate, are required and optimal conditions are the same for both. This suggests that a-ketoglutarate is the first acceptor of the oxygen and that a peroxy compound, perhaps enzyme bound persuccinate, then attacks the 4-trans position of proline to yield the intermediate shown above. Gleavage of the peroxy compound would then yield equal amounts of succinate and hydroxyproline. 

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