TABLE 2.

Kinetic constants obtained from spectrophotometric coupled-enzyme assays

Enzymekcat (s−1)kcat/Km (M−1 s−1)
SgaEa253.8 × 104
SgbEa121.7 × 104
SgaHb517.7 × 104
SgaUc2.95.5 × 103
YiaKd1101.4 × 105
LyxKe (3-keto-l-gulonate)488.9 × 104
LyxKe (l-xylulose)1201.1 × 105
SgbHb641.0 × 105
  • a The assay mixtures (1 ml at 25°C) contained l-ribulose 5-phosphate (0.1 to 10 mM), 50 mM glycoaldehyde, 0.1 mM thiamine pyrophosphate, 50 mM K+HEPES (pH 7.5), 10 mM MgCl2, 10 U of α-glycerophosphate dehydrogenase, 1 U of transketolase, 45 U of triose phosphate isomerase per ml, and 0.16 mM NADH. The change in absorbance at 340 nm was quantitated.

  • b The assay mixtures (1 ml at 25°C) contained β-keto-l-gulonate 6-phosphate (0.2 to 2.9 mM), 50 mM K+HEPES (pH 7.5), and 10 mM MgCl2. The enzyme was removed by centrifugation through a Biomax-10 (10,000 Da) Millipore centrifugal filter device. After incubation of an aliquot (800 μl) with 30 U of calf intestine alkaline phosphatase at 37°C for 5 min, 300 μl of a 5% solution of ZnSO4 · 7H2O and 300 μl of 0.15 M Ba(OH)2 were added; the precipitate was removed by centrifugation. An aliquot (800 μl) of the supernatant was assayed for l-xylulose by reaction with l-cysteine and carbazole (17). The absorbance at 540 nm was measured; the molar extinction coefficient for the chromophore was determined to be 1,367 M−1 cm−1. A stock solution of β-keto-l-gulonate 6-phosphate was generated by incubating 3.25 mM l-diketogulonate, 10 mM MgCl2, 6 mM ATP, 60 U of YiaK per ml, 50 U of LyxK per ml, and a molar equivalent of NADH for 20 min at 25°C.

  • c The assays (1 ml at 25°C) contained l-xylulose (0.2 to 16.7 mM), 1.5 mM ATP, 15 mM acetyl phosphate, 50 mM glycoaldehyde, 0.1 mM thiamine pyrophosphate, 50 mM K+HEPES (pH 7.5), 10 mM MgCl2, 22 U of acetate kinase, 110 U of LyxK, 10 U of α-glycerophosphate dehydrogenase, 1 U of transketolase, 45 U of a triose phosphate isomerase, 5 U of SgaE per ml, and 0.16 mM NADH. The change in absorbance at 340 nm was quantitated. The assays were incubated for 30 min at 25°C to generate l-xylulose 5-phosphate prior to addition of SgaU.

  • d The assay mixtures (1 ml at 25°C) contained l-diketogulonate (0.050 to 3.4 mM), 50 mM Tris-HCl (pH 7.5), and 0.16 mM NADH. The change in absorbance at 340 nm was quantitated.

  • e The assay mixtures (1 ml at 25°C) contained 3-keto-l-gulonate (0.08 to 9.2 mM; prepared in situ with YiaK) or l-xylulose (0.066 to 10.7 mM), 1.5 mM phosphoenolpyruvate, 1.5 mM ATP, 50 mM K+HEPES (pH 7.5), 10 mM MgCl2, 9 U of pyruvate kinase, 9 U of lactate dehydrogenase per ml, and 0.16 mM NADH. The change in absorbance at 340 nm was quantitated.