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Probing the determinants of the metal binding specificity in HDAC8 (768.7)
Author(s) -
Kim Byungchul
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.768.7
Subject(s) - chemistry , dissociation constant , binding site , stereochemistry , cofactor , active site , histidine , metal , enzyme , biochemistry , receptor , organic chemistry
HDACs catalyze the hydrolysis of ε‐N‐acetyl lysine residues on proteins acetylated by histone acetyltransferases. Upon their discovery, this enzyme was identified as Zn2+‐dependent metalloenzymes. However, current activity and binding affinity studies suggest that Fe2+ could be the native metal cofactor in vivo. This has a number of biologically relevant implications, including 1) metal switching regulatory mechanism, 2) substrate specificity based on the identity of metal status. Therefore I am presenting the determinants of metal selectivity in HDAC8 in vitro to provide insight into the intracellular metal specificity in vivo. In HDAC8, the second shell of amino acid surrounding the metal coordinating side chains is predominantly hydrophobic. This is contrary to many prototypical metalloenzymes, such as carbonic anhydrase II (CAII). The lack of second shell hydrogen bonds may be an important factor contributing to the weaker Zn2+ affinity of HDAC8 compared to CAII, allowing facile switching between Zn2+ and Fe2+ cofactors. To investigate these structural determinants, I have prepared HDAC8 mutants and I am measuring the metal ion affinity and dissociation rate constants of wild‐type and mutants. Surprisingly the dissociation rate constants for Zn2+ and Fe2+ from the HDAC8‐metal complex in mutants are comparable despite the large difference in binding affinity. Previous studies indicate that binding to the 1st K+ site inhibits the activity, while binding to the second K+ site activates catalysis possibly through structural stabilization. This indicates that K+ binding to two different sites may affect the Zn2+ binding site. To study this, I am measuring the dissociation rate constant for zinc and iron binding to HDAC8 at varying concentrations of monovalent cation (K+ and Na+). The Zn2+ dissociation rate constant from metal‐bound HDAC8 decreases as the concentration of monovalent cation (K+/Na+) is increased. Grant Funding Source : Supported by NIH's NIGMS 2RO1GM040602‐22