Essential groups at the active site of Trapaeolum invertase

Amino acid residues involved at the active site of Tropaeolum majus invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were determined. Photo-oxidation and Woodward's reagent K revealed the presence of histidyl and carboxylic groups at the active site of the enzyme. These groups were protected by sucrose and trehalose but not by fructose. Thiol groups were determined with sodium p-chloromercuribenzoate and with 5,5'-dithio-bis(2-nitrobenzoic acid). These groups were protected by fructose and to a lesser degree by sucrose, but were not protected by trehalose. The pH dependence of V-max and K-m at 27 degrees and 37 degrees is consistent with the presence of carboxyl and histidyl residues at the invertase active site. The pK(a) values obtained correspond to ionization heats of 1700 cal mol(-1) and 6820 cal mol(-1). The results were consistent with the presence of imidazole, carboxylic and thiol groups at the active site. (C) 1998 Elsevier Science Ltd. All rights reserved.