RECOGNITION OF “MINIMAL” LIGANDS BY ENOLASE SUPERFAMILY ENZYMES

Abstract

Enolase superfamily (ENS) enzymes share a common half-reaction, the Mg2+-dependent, Brønsted base-catalyzed abstraction of an α-proton from a carboxylic acid substrate to form an enolate. The active site architecture of the ENS enzymes L-talarate/galactarate dehydratase (TGD), D-(-)-tartrate dehydratase (TarD), and β-methylaspartate ammonia-lyase (MAL) were probed using the “minimal” ligands, tartronate and 3-hydroxypyruvate (3-HP). Both were weak competitive inhibitors of TarD (Km = 27 ± 7 μM) with Ki values of 500 ± 100 μM and 1800 ± 400 μM, respectively, and weak inhibitors of TGD (Km = 900 ± 200 μM) with IC50 values of 17 ± 1 mM and 15 ± 1 mM, respectively. MAL (Km = 980 ± 160 μM) bound tartronate weakly, but bound 3-HP as a good competitive inhibitor (Ki = 1800 ± 200 μM). Mass spectrometry experiments revealed that 3-HP forms a Schiff base with the catalytic Lys in the active sites of all three enzymes, which is not deprotonated as it is with MR. Investigation of the metal ion binding site of TarD revealed that 0.5 M EDTA did not obviate activity as it does for MR.