Characteristics and specificity of purified n-feruloylglycine amidohydrolase from isolated barley embryos

Abstract

N-Feruloylglycine amidohydrolase with an estimated M r of 155 000, showed with N-feruloylglycine at pH 8 and 30° a K m of 85 μM, a V m of 3.92 nmol/0.1 mg protein/min, a physiological efficiency ( V m / K m ) of 46.1.10 3 and an apparent activation energy of 43.5 kJ/mol. Sulphydryl reagents were shown to decrease enzyme activity and relative high concentrations (10 mM) of metal chelating reagents (EDTA, dithizone and o-phenanthroline) were also inhibitory. The effect produced by o-phenanthroline could be partially reversed by CuCl 2, and CoCl 2, and to a lesser extent by ZnCl 2 and MnCl 2. N-Feruloyldipeptides such as N-feruloylglycyl- l-phenylalanine ( K i = 42 μM; α = 7.8 and β = O) and N-feruloylglycyl- l-leucine ( K i = 300 μM; α = 5.8; β = O) were potent reversible mixed type inhibitors. The purified enzyme did not show any deformylase or amidase activity and was thus totally different from N-acylamino acid amidohydrolase (EC 3.5.1.14). In addition, the pseudopeptide bond of N-feruloylglycine was not split by a series of peptidases and proteinases. N-Feruloylglycine amidohydrolase is a new acylase of plant origin and the name proposed for the enzyme is well supported by substrate specificity studies.