The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation.

The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation.
J Inorg Biochem. 2016 Oct 17;166:26-33
Authors: Hangasky JA, Taabazuing CY, Martin CB, Eron SJ, Knapp MJ
Abstract
The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O2 via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn(803) residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O2 activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp(201)) of FIH linked substrate binding and O2 binding sites. Asp(201) variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated O2 with a catalytic efficiency similar to that of wild-type (WT) FIH (kcataKM(O2)=0.17μM(-1)min(-1)), but led to defects in the coupling of O2 activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (kcat/KM(CTAD)=0.42μM(-1)min(-1)) and D201G (kcat/KM(CTAD)=0.34μM(-1)m...

Source: Journal of Inorganic Biochemistry - October 16, 2016 Category: Biochemistry Authors: Hangasky JA, Taabazuing CY, Martin CB, Eron SJ, Knapp MJ Tags: J Inorg Biochem Source Type: research