4. CONCLUSIONS
The two-step lipoylation process of H protein catalyzed by E. coli LplA has been kinetically studied as a whole and in individual steps for the first time in this work. The experimental results revealed a highly asymmetric kinetic behavior and hitherto unreported phenomena of H protein lipoylation: the first adenylation step is faster than the second lipoate transfer step by 2-3 orders of magnitude, leading to a high accumulation of the intermediate Lip-AMP under in vitroassay conditions. Unexpectedly, the second step was activated not only by the substrate Hapo but also by the product Hlip and the mutant HK64A lacking the binding site of the lipoate. Moreover, a competitive Lip-AMP hydrolysis side-reaction was found which is also activated by the different forms of H-proteins. These new experimental observations suggest that there may be local conformational changes in the Lip-AMP binding pocket of LplA induced by the interactions and binding of H protein and its variants. Lip-AMP is then activated and attacked by either the amino group of the lysine residue of H protein or by the hydroxyl group of H2O competitively. Structural and molecular dynamic studies of interactions of LplA and H protein could shed more light into H protein lipoylation, an essential process of several multiple enzyme systems with high impacts in biomedicine and biotechnology.