3.1 Activation of H protein lipoylation
Previously, H protein lipoylation catalyzed by LplA was only studied in
a low concentration ranges of Hapo (up to about 5 µM)
due to the limitation of the isotope radioactive labeling method used
for the activity assay (Fujiwara et al., 2005). Here, we used our newly
developed HPLC methods (see Materials and Methods) to quantitatively
analyze the rate of H protein lipoylation in a wide range of
concentrations of Hapo. At the same time, we also
examined the possible effects of Hlip as the reaction
product on the lipoylation process. Previous in vivo study of the
lipoylation process in E. coli revealed a limited lipoylation of
H protein and implied a possible inhibition of H protein lipoylation by
its final product Hlip at low expression of LplA (Zhang
et al., 2019). Unexpectedly, in this study, both Hapo as
the reaction substrate and Hlip as the reaction product
showed positive effects on the lipoylation process (Fig. 2a-f).
Fig. 2a shows that the effect of Hapo concentration on
Hlip formation is significant even in a short reaction
time of 15 min. A reaction time of 5-15 min and a Hapoconcentration in the range of 0.19-4.5 µM were previously used in
literature for the activity assay of LplA and the determination of the
km value of Hapo (Fujiwara et al.,
2005). In this range we also observed very low lipoylation of
Hapo. In our study, when higher initial concentration of
Hapo was used, the concentration of the formed
Hlip significantly increased. In fact, it increased with
the increase of the initial Hapo concentration up to 200
μM (Fig. 2a). As shown in Fig. 2b for two starting concentrations (10 µM
and 100 µM, respectively) of Hapo, the concentration of
Hlip reached maximum with a nearly 100% of lipoylation
only after about 5 to 15 hours under the experimental conditions. From
the reaction curves as shown in Fig. 2a we can calculate the initial
reaction rate (v lip) of Hlip as a
function of initial Hapo concentration (Fig. 2c). It
turned out that v lip does not follow a typical
Michaelis-Menten kinetics as implied in previous studies (Fujiwara et
al., 2005). Instead, it shows first a pseudo-saturation up to an initial
Hapo concentration of about 10-50 µM, but increases
again when the initial Hapo concentration was higher
than 50 µM. Because the lipoylation product Hlip also
significantly affect v lip (Fig. 2d-f), an exact
estimation of the km value even for this
pseudo-saturation is not possible. Neglecting the effects of both
Hapo and Hlip as activators resulted in
an apparent km value of less than 3 µM (assuming a
saturation at about 10 µM Hapo). This is in the same
range of 1.2 µM as determined by Fujiwara et al. (Fujiwara et
al., 2005). These authors also determined a v maxof 0.26 µmol/min/mg Protein for E. coli LplA. This is comparable
to a v lip value at a Hapoconcentration of about 8 µM in Fig. 2c. In our assayv lip increased significantly when the initial
Hapo was beyond 50 µM. Additional measurements with LplA
collected from a new batch of protein expression and purification showed
that v lip increased nearly linearly in a
Hapo concentration range of 50 - 400 µM (data not
shown). These results indicated that Hapo may act not
only as a substrate, but also as an activator for its own lipoylation.
Surprisingly, Hlip also acts as an activator of H
protein lipoylation (Fig. 2d and 2e). The relationship between the
externally added initial Hlip concentration and the
initial reaction rate v lip showed a pattern
similar to that between Hapo andv lip (Fig. 2f). Hlip can active
the lipoylation process, rather than cause a product inhibition. In
general, it can be stated that Hlip causes a positive
feedback activation of the lipoylation process.
To further examine the effects of different forms of H protein on the
lipoylation of Hapo, we constructed a mutant protein of
Hapo with mutation of the key residue lysine64 into
alanine (HK64A). The lysine residue at the position 64
in Hapo is the active site for the binding of lipoic
acid. When it is mutated to alanine, the derived Hapomutant should not participate in the lipoylation as a substrate. As
shown in Fig. 3a, HK64A did slightly promoted the
lipoylation of H protein, but the activation was much less significant
compared to those of Hapo and Hlip. It
is noted that Hapo has the most significant effect on
the formation of Hlip. This might be due to the fact
that Hapo is both a substrate and an activator.