Mechanistic Studies on Prolyl-4-Hydroxylase: Demonstration That the Ferryl Intermediate Does Not Exchange with Water

Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. In contrast to deacetoxy/deacetylcephalosporin C synthase, p-hydroxyphenylpyruvate hydroxylase, lysyl hydroxylase and α-ketoisocaproate oxygenase, no incorporation of 18O-labeled water into the hydroxylated product was found for the human type I prolyl-4-hydroxylase when N-Cbz-Gly-L-Phe-L-Pro-Gly-OEt was used as a substrate. This suggests that the ferryl intermediate for this enzyme is not solvent accessible.