COMPLEMENTATION ANALYSIS OF MUTANTS OF 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE REVEALS THE ENZYME IS A DIMER WITH SHARED ACTIVE-SITES

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxy late synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in t he ethylene biosynthetic pathway. ACS shares the conservation of 11 in variant residues with a family of aminotransferases that includes aspa rtate aminotransferase. Site-directed mutagenesis on two of these resi dues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly redu ces enzymatic activity, indicating their importance in catalysis. Thes e mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutan t is coexpressed with the K278A mutant protein, there is partial resto ration of enzyme activity, suggesting that the mutant proteins can dim erize and form active heterodimers. Coexpressing a double mutant with the wildtype protein reduces wild-type activity, indicating that inact ive heterodimers are formed between the wild-type and the double mutan t protein subunits. Furthermore, hybrid complementation shows that ano ther tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-A CS4 have limited capacity for heterodimerization. The data suggest tha t ACS functions as a dimer with shared active sites.