Site-Directed Mutagenesis of Residues in a Conserved Region of Bovine Aspartyl (Asparaginyl) β-Hydroxylase: Evidence That Histidine 675 Has a Role in Binding Fe2+

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Abstract
The roles in catalysis of several residues in bovine aspartyl (asparaginyl) β-hydroxylase that are located in a region of homology among α-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for α-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) β-hydroxylase and, by analogy, other α-ketoglutarate-dependent dioxygenases.