EXPLORING CATALYSIS IN THE MANDELATE RACEMASE SUBGROUP OF THE ENOLASE SUPERFAMILY: SUBTLE DIFFERENCES IN THE CATALYTIC MACHINERY

Abstract

The mandelate racemase (MR) subgroup of the enolase superfamily of enzymes (ENS) is mechanistically diverse but structurally similar. This work focused on conserved active site residues in subgroup members MR and L-fuconate dehydratase (FucD). The impact of a Tyr 137-Lys 164-Lys 166 triad on the pKa of Lys 166 of MR was investigated. A Y137F mutation increased the pKa of both Lys 166 and His 297 by 1-1.4 pKa units. FucD was assayed for inhibition with tartronate and 3-hydroxypyruvate (3-HP), which bind the conserved Brønsted acid-base catalysts of MR. Tartronate was found to be a weak linear mixed-type inhibitor of FucD (Ki = 8.37 ± 0.72 mM, α = 7.53 ± 1.16) and 3-HP irreversibly inactivated FucD (kinact = 1.49 ± 0.0058 x 10-4 s-1, KI = 7.94 ± 0.24 mM, kinact/KI = 0.0187 ± 0.0007 M-1s-1). Lastly, Trp 101 on the interdigitating loop of FucD was found to be critical for catalysis.