Biochemical properties of 1-aminocyclopropane-1-carboxylateN-malonyltransferase activity from early growing embryonic axes of chick-pea (Cicer arietinumL.) seeds

Abstract

In the present work, certain biochemical characteristics of the enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase (ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate (ACC) in chickpea (Cicer arietinum) are described. Phosphate buffer was the most appropriate buffer with regard to enzyme stability and, therefore, ACC N-MTase was extracted, assayed and purified in the presence of this buffer. ACC N-MTase was partially purified approximately 900-fold from embryonic axes of chick-pea seeds using ammonium sulphate precipitation, hydrophobic interaction and molecular filtration chromatography. By gel filtration chromatography on Superose-12, the molecular mass of the enzyme was estimated to be 54±4 kDa. ACC N-MTase had an optimal pH and temperature of 7.5 and 40°C, respectively, as well as a K m for ACC and malonyl-CoA of 400 μM and 90 μM, respectively. D-Phenylalanine was a competitive inhibitor of ACC N-MTase with respect to ACC (K i of 720 μM), whereas co-enzyme A was a competitive product inhibitor with respect to malonyl-CoA (K i of 300 μM) and a non-competitive inhibitor with respect to ACC (K i of 600 μM). Under optimal assay conditions, ACC N-MTase was strongly inhibited by (a) divalent [Zn 2+ > Mg 2+ > Cu 2+ >> Co 2+ > (NH 4 ) 2+ > Fe 2+ ] and monovalent metal cations (Li + > Na + > K + ), without activity being detected in the presence of Hg 2+ , and (b) PCMB or mersalic acid, suggesting that sulphydryl group(s) are involved at the active site of the enzyme.