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Metabolism and Enzymology

Purification and Characterization of 1-Aminocyclopropane-1-Carboxylate N-Malonyltransferase from Etiolated Mung Bean Hypocotyls ¹

Department of Horticulture, The Pennsylvania State University, University Park, Pennsylvania 16802, Department of Molecular and Cellular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, Shanghai Institute of Plant Physiology, Academia Sinica, 300 Fenglin Road, Shanghai, China 200032

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg^–1 protein h^–1. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent K_m of 0.5 mM for ACC and 0.2 mM for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol^–1 degree^–1.