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-amino-cyclopropane-1-car boxylic 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 a cids at the C terminus was chosen as the control enzyme for the study of th e function of R286 in ACC synthase. R286 of the tomato ACC synthase was mut ated to a leucine via site-directed mutagenesis. The ACC synthase mutant R2 86L was purified using a simplified two-step purification protocol. Circula r dichroism (CD) analysis indicated that the overall three-dimensional stru cture of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC s ynthase for its cofactor PLP was reduced 20- to 25-fold compared with contr ol. Kinetic analysis of R286L showed that this mutant ACC synthase had a si gnificantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an i ncreased K-m of 730 mu M for AdoMet, leading to an 8,000-fold decrease in o verall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.