The 2-oxoglutarate Binding Site of Prolyl 4-hydroxylase. Identification of Distinct Subsites and Evidence for 2-oxoglutarate Decarboxylation in a Ligand Reaction at the Enzyme-Bound Ferrous Ion

Eur J Biochem. 1984 Jan 16;138(2):239-45. doi: 10.1111/j.1432-1033.1984.tb07907.x.

Abstract

The structure and function of the 2-oxoglutarate binding site of prolyl 4-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 Fe2+, the Ki values ranging from 0.8 microM 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 microM and 2 microM, 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 II consists of two cis-positioned equatorial coordination sites of the enzyme-bound ferrous ion and is chelated by the C1-C2 moiety, while subsite III 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / drug effects
  • Binding, Competitive
  • Chick Embryo
  • Decarboxylation
  • Ferrous Compounds / metabolism*
  • Iron / metabolism*
  • Ketoglutaric Acids / metabolism*
  • Ligands
  • Procollagen-Proline Dioxygenase / metabolism*
  • Protein Binding

Substances

  • Ferrous Compounds
  • Ketoglutaric Acids
  • Ligands
  • Iron
  • Procollagen-Proline Dioxygenase