Procollagen-proline dioxygenase, commonly known as prolyl hydroxylase, is a member of the class of enzymes known as 2-oxoglutarate-dependent dioxygenases. These enzymes catalyze the incorporation of oxygen into organic substrates through a mechanism that requires 2-oxoglutarate, Fe²⁺, and ascorbate. This particular enzyme catalyzes the formation of (2S, 4R)-4-hydroxyproline, a compound that represents the most prevalent post-translational modification in the human proteome.

Procollagen-proline dioxygenase catalyzes the following reaction:

procollagen L-proline + 2-oxoglutarate + O₂ → (2S, 4R)-4-hydroxyproline + succinate + CO₂

The mechanism for the reaction is similar to that of other dioxygenases, and occurs in two distinct stages: In the first, a highly reactive Fe(IV)=O species is produced. Molecular oxygen is bound end-on in an axial position, producing a dioxygen unit. Nucleophilic attack on C2 generates a tetrahedral intermediate, with loss of the double bond in the dioxygen unit and bonds to iron and the alpha carbon of 2-oxoglutarate. Subsequent elimination of CO₂ coincides with the formation of the Fe(IV)=O species. The second stage involves the abstraction of the pro-R hydrogen atom from C-4 of the proline substrate followed by radical combination, which yields hydroxyproline.

As a consequence of the reaction mechanism, one molecule of 2-oxoglutarate is decarboxylated, forming succinate. This succinate is hydrolyzed and replaced with another 2-oxoglutarate after each reaction, and it has been concluded that in the presence of 2-oxoglutarate, enzyme-bound Fe²⁺ is rapidly converted to Fe³⁺, leading to inactivation of the enzyme. Ascorbate is utilized as a cofactor to reduce Fe³⁺ back to Fe²⁺.

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