The structure and function of the 2-oxoglutarate binding site of prolyl4-hydroxylase was studied by assaying the inhibitory potential of 24 selected aliphatic or aromatic compounds. All except one of them inhibited the enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fez+, the Ki values ranging from 0.8 pM to over 15 mM. The Ki values for the two most effective inhibitors, pyridine 2,5-dicarboxylate and 2,4-dicarboxylate, were about 0.8 pM and 2 pM, these compounds being the most potent inhibitors of prolyl 4-hydroxylase with respect to 2-oxoglutarate known so far. Only one of the compounds tested, 2-oxoadipinate, was able to support hydroxylation by replacing 2-oxoglutarate as a cosubstrate. The data suggest that the 2-oxoglutarate binding site can be divided into three distinct subsites. Subsite I is probably a positively charged side chain of the enzyme that ionically binds the C5 carboxyl group of the 2-oxoglutarate, subsite I1 consists of two cis-positioned equatorial coordination sites of the enzyme-bound ferrous ion and is chelated by the C1-C2 moiety, while subsite I11 involves a hydrophobic binding site in the C3 -C4 region of the cosubstrate. The sp3 rehybridization of C2 within the chelating moiety of the cosubstrate appears to be a crucial event during decarboxylation that proceeds in the form of a ligand reaction inside the Fe2+ coordination sphere.Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of certain proline residues in peptide linkages (for reviews, see [l-31). The minimum sequence requirement for hydroxylation is fulfilled by an -Xaa-Pro-Gly-triplet, and the reaction requires ferrous ions, 2-oxoglutarate, molecular oxygen and ascorbate. The 2-oxoglutarate is stoichiometrically decarboxylated, one atom of the O2 molecule being incorporated into the succinate while the other is incorporated into the hydroxyl group [l -31. The results of extensive kinetic studies [4-61 and other data [7, 81 are consistent with an ordered binding of Fe2 +, 2-oxoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, C02, succinate, and Fez+, in which Fe2 + does not leave the enzyme between the majority of the catalytic cycles and in which the order of release of the hydroxylated peptide and CO, is uncertain. Ascorbate is not consumed stoichiometrically [5, 91, and pure enzyme preparations can catalyze hydroxylation for a number of catalytic cycles in the complete absence of this vitamin [6, 81. These findings, together with the kinetic [6,10) and other data [S], suggest that ascorbate is required to prevent oxidation of the enzyme-bound iron and possibly some other groups on the enzyme molecule between some catalytic cycles, but not the majority.A detailed stereochemical mechanism has recently been suggested for prolyl4-hydroxylase [ll], in which it is proposed that binding of 2-oxoglutarate (compound...