Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase

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Abstract
Prolyl 4‐hydroxylase (EC 1.14.11.2), an α2β2 tetramer, catalyzes the formation of 4‐hydroxyproline in collagens. We converted 16 residues in the human α subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild‐type β subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the Km for Fe2+ 15‐fold and that for 2‐oxoglutarate 5‐fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the Km for 2‐oxoglutarate 15‐fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85‐95%; all these mutations increased the Km for 2‐oxoglutarate 2‐ to 3‐fold and enhanced the rate of uncoupled decarboxylation of 2‐oxoglutarate as a percentage of the rate of the complete reaction up to 12‐fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C‐5 carboxyl group of 2‐oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C‐1 carboxyl group of 2‐oxoglutarate and the decarboxylation of this cosubstrate.