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United States Department of Agriculture

Agricultural Research Service

Title: Complementation Analysis of Mutants of 1-Aminocyclopropane1-Carboxylate Synthase Reveals the Enzyme Is a Dimer with Shared Active Sites

item Tarun, Alice - USDA/UCB PGEC
item Theologis, Athanasios

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 15, 1998
Publication Date: May 15, 1998
Citation: Tarun, A., Theologis, A. 1998. Complementation analysis of mutants of 1-aminocyclopropane1-carboxylate synthase reveals the enzyme is a dimer with shared active sites. Journal of Biological Chemistry, 273(20) 12509-12514.

Interpretive Summary: The data suggest that ACS functions as a dimer with shared active sites.

Technical Abstract: The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 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.

Last Modified: 4/18/2015
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