The Multiple Roles of Conserved Arginine 286 of 1-Aminocyclopropane-1-Carboxylate Synthase. Coenzyme Binding, Substrate Binding, and Beyond1
AUTOR(ES)
Zhou, Huiqing
FONTE
American Society of Plant Physiologists
RESUMO
A pyridoxal 5′-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-l-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-l-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (kcat) of 8.2 × 10−3 s−1 and an increased Km of 730 μm for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.
ACESSO AO ARTIGO
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