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