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.

Subjects

H protein

hydrolysis

lipoate–protein ligase A

lipoylation

protein interaction

Funding(s)

Teilprojekt von SPP 1934: Multiskalige modellgestützte Untersuchungen funktionaler Enzym- und Proteinagglomerate für biotechnologische Anwendung - Teil 2: Von der Struktur zur Funktion

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