2.5 Analysis of the overall reaction and the individual reaction
steps of lipoylation
- Overall reaction activity. The overall reaction activity was
determined at constant concentrations of R-(+)-lipoic acid (2 mM), ATP
(2 mM), MgCl2 (2 mM), Hapo (0.5 -400
μM), and LplA (10 μM) The reaction was started with the addition of
ATP. The amount of lipoylated H protein in the mixture was determined
using HPLC as described in our previous study (Zhang et al., 2019).
- Activity assay of the lipoate adenylation reaction (Step 1).The reaction activity was determined in a reaction mixture of 0.25 mL
containing 10 μM LplA, 2 mM ATP, 2 mM MgCl2, and 2 mM
R-(+)-lipoic acid. The final volume was made up with 50 mM Tris-HCl
(pH 7.5). The reaction was initiated by adding ATP, incubated at 30 ºC
for 15 min (unless stated otherwise), and terminated by heating for
90s in boiling water to completely denature and precipitate LplA.
Lip-AMP was chromatographically separated and quantified as described
previously (Fujiwara et al., 2010), using a gradient HPLC on a
Shimadzu Shim pack GIST C18 column (5 µm, 4.5 × 150 mm), with the
mobile phase A being acetonitrile containing 0.1% trifluoroacetic
acid, and the mobile phase B being 50 mM PBS buffer (pH 6.6)
containing 5 mM tetrabutylammonium hydrogen sulfate (TBAHS). The
formation of Lip-AMP can be observed as a single, clearly resolved
peak. Because no chemical standard of Lip-AMP is commercially
available, Lip-AMP was quantified indirectly as follows: after ATP,
ADP and AMP were quantitatively determined, Lip-AMP is calculated as
the difference between the initial amount of ATP and the total amount
of the ATP remained and the ADP and AMP formed.
- Activity assay of the lipoate transfer reaction (Step 2). The
determination of the second step activity was similar to that in the
first step, in which lipoic acid, ATP and MgCl2 were
replaced with Lip-AMP and Hapo. Lip-AMP was obtained
through an overnight reaction of the first step and after a heat
treatment of the reaction mixture in boiling water for 1 minute to
denature LplA completely. Then, the sample was centrifuged at 10,000 x
g and 4 °C for 1 minute. The supernatant was collected and used as the
substrate of the second step. The lipoate transfer reaction was
initiated by adding LplA, incubated at 30 ºC for 15 min, and
terminated by heating 1 minute in boiling water. The method for the
detection of Hlip was the same as that used for the
overall reaction.