Poster abstracts
Poster number 11 submitted by Jenna Greve
Coordination chemistry and catalytic activity of the Human Aspartyl (Asparaginyl) Hydroxylase
Jenna M. Greve (OSBP The Ohio State University), Andrew M. Pinkham (Department of Chemistry and Biochemistry The Ohio State University), James A. Cowan (Department of Chemistry and Biochemistry The Ohio State University)
Abstract:
Human Aspartyl Asparaginyl Beta-Hydroxylase (HAAH) is a member of a superfamily of nonheme iron α-ketoglutarate (αKG) dependent oxygenase enzymes that catalyze a variety of metabolically important transformations. The catalytically active isoform of HAAH is overexpressed in 95% of tested cancers, leading to questions about its role in malignant transformations. HAAH is known to hydroxylate aspartate and asparagine residues in specific epidermal growth factor (EGF) like domains. The 2-histidine-1-carboxylate iron binding triad is a highly conserved feature of the active site of αKG dependent oxygenase enzymes, however, HAAH crystal structures show a nickel or manganese center to be ligated by only two histidine residues. We aim to explore the primary enzyme chemistry to compare to known family members so we can lead into deeper questions about the role of hydroxylation by HAAH in normal cellular function and cancer. To investigate the active site coordination and catalytic activity we utilize a variety of techniques including Isothermal Titration Calorimetry and LC/MS based activity assays. We show an entropically driven binding with little difference in metal binding affinity for a variety of active site mutations with potential coordination activity, verifying the 2 histidine active site. A third mutant shows no apparent metal binding by ITC and no enzymatic activity but is too far from the active site iron to be directly coordinating. These results expand the known active site designs of αKG dependent dioxygenases and indicate the canonical carboxylate is not necessary for enzymatic activity. Investigation of the coordination chemistry and catalytic activity will allow us to further probe the role of HAAH hydroxylation in malignant phenotypes as well as normal cellular activity.
Keywords: enzyme activity, hydroxylase, active site coordination