Zhang, XinyiXinyiZhangNie, JingleiJingleiNieZheng, YuanminYuanminZhengRen, JieJieRenZeng, An-PingAn-PingZeng2020-10-302020-10-302020-12Biotechnology and Bioengineering 117 (12): 3677-3687 (2020-12)http://hdl.handle.net/11420/7725Protein 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.en0006-359220201236773687H proteinhydrolysislipoate–protein ligase Alipoylationprotein interactionJournal Article10.1002/bit.2752632749694Journal Article