SITE-DIRECTED MUTAGENESIS OF RESIDUES IN A CONSERVED REGION OF BOVINEASPARTYL (ASPARAGINYL) BETA-HYDROXYLASE - EVIDENCE THAT HISTIDINE-675WAS A ROLE IN BINDING FE2+

The roles in catalysis of several residues in bovine aspartyl (asparag inyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using sit e-directed mutagenesis. Previous studies have shown that when histidin e 675, an invariant residue located in this highly conserved region, w as mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has b een undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replac ements for histidine 675 correlates with the potential to coordinate m etals and not with size, charge, or hydrophobic character. Furthermore , the increase in K-m for Fe2+ observed with the H675D and H675E mutan t enzymes can account for their partial activities relative to wild ty pe. No significant changes in the K-m for alpha-ketoglutarate (at satu rating Fe2+) or V-max were observed for these mutants. These results s upport the conclusion that histidine 675 is specifically involved in F e2+ coordination. Further site-directed mutagenesis of other highly co nserved residues in the vicinity of position 675 demonstrates the impo rtance of this region of homology in catalysis for Asp (Asn) beta-hydr oxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygena ses.