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: (1) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate Lip-AMP; (2) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (3) both the lipoylation of H protein and its hydrolysis are 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.