Xinyi Zhang, J. Nie, Yuanmin Zheng
Aug 4, 2020
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Influential Citations
6
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Journal
Biotechnology and Bioengineering
Abstract
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.