A continuous assay for l-talarate/galactarate dehydratase using circular dichroism

Abstract

l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ± 2 deg cm2 dmol−1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters Km, kcat, and kcat/Km were 0.38 ± 0.05 mM, 4.8 ± 0.1 s−1, and 1.3 (±0.2) × 104 M−1s−1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry46, 9564–9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (Ki = 10.7 ± 0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.