Pyruvate kinase lactate dehydrogenase

For kinetic
measurements of purified VanX, a 200 μL reaction containing
50 mM deuterated-Tris, pH 7.9, 10 mM MgCl₂, 1 μM
VanX, and 10 mM d-Ala-d-Ala was prepared in ddH₂O and incubated at 37 °C for 16 h (a control reaction
was also prepared without VanX). Reactions were lyophilized for 24
h and resuspended in 800 μL of D₂O at pD 7.9. ¹H NMR spectra were recorded with a Unity INOVA 500NB instrument
and analyzed with NUTS software.
γ-Glutamyl amide synthetase
activity of Csal_0679 was assayed by measuring formation of ADP from
ATP, where production of ADP was followed by the decrease of absorbance
of NADH at 340 nm at 25 °C due to oxidation of NADH via substrate/product
coupled pyruvate kinase and lactate dehydrogenase. The reaction mixture
contained variable concentrations of tested substrate and defined
concentration of co-substrate, 50 mM Tris-HCl buffer (pH 7.9), 10
mM KCl, 15 mM MgCl₂, 5 mM ATP, 2.5 mM PEP, 0.16 mM NADH,
8 units of pyruvate kinase/lactate dehydrogenase from rabbit muscle
(Sigma-Aldrich), and 1.2 × 10^–6 M Csal_0679
in a final volume of 200 μL. Measurements of kinetic parameters
for glutamate were made at 20 mM ethanolamine or l-alaninol,
respectively. Kinetic constants for ethanolamine or l-alaninol
were measured at 50 mM glutamate.