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

作者：Easton Nicole M; Aboushawareb Sarah A E; Bearne Stephen L^*

来源：Analytical Biochemistry, 2018, 544: 80-86.

DOI：10.1016/j.ab.2017.12.015

• Dehydratase
• Assay
• Circular dichroism
• Kinetics
• m-Galactarate
• 5-Keto-4-deoxy-D-glucarate

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 ([theta](323)) for the 5-KDG formed was determined to be 202 +/- 2 deg cm(2) dmol(-1), which was used to convert observed rates (Delta theta/Delta t) into concentration-dependent rates (Delta c/Delta t). The kinetic parameters K-m, k(cat), and k(cat)/K-m were 0.38 +/- 0.05 mM, 4.8 +/- 0.1 s(-1), and 1.3 (+/- 0.2) x 10(4) M(-1)s(-1), respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry 46, 9564-9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (K-i; = 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.

• 出版日期2018-3-1