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.