Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate–protein ligase A

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two‐step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate–protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl‐AMP (Lip‐AMP); (b) Lip‐AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles. Protein lipoylation is essential for the function of many key enzymes, but barely studied kinetically. We discovered that the multifunctional enzyme lipoate‐protein ligase A (LpLA) catalyzing a two‐step reaction cascade of H protein lipoylation exhibits unusual kinetics: the speed of the first reaction being much faster than the second one which results in accumulation and hydrolysis of the intermediate Lip‐AMP. LpLA undergoes conformational changes upon interaction with H‐protein, leading to accelerated lipoylation and hydrolysis.