The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond.
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 (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM 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.
Zhou
H
H
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Special Administrative Region, The People's Republic of China.
Wang
H W
HW
Zhu
K
K
Sui
S F
SF
Xu
P
P
Yang
S F
SF
Li
N
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D007700:Kinetics; D007930:Leucine; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013552:Swine
D011994:Recombinant Proteins; D001120:Arginine; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase; D007930:Leucine