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-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These 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 mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.
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Plant Gene Expression Center, Albany, California 94710, USA.
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D001665:Binding Sites; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase