Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases) but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the Glc-Fru linkage in sucrose while plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the 3-D structures of Arabidopsis thaliana cell wall invertase 1 (AtcwINV1) and chicory 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether sucrose or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this manuscript, we present site-directed mutagenesis based data on AtcwINV1 showing that the Asp239 residue fulfills an important role in both binding and hydrolysis of sucrose. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of sucrose. Surprisingly, a D239A mutant acted as a 1-FEH preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by few mutational changes. In general, family 32 and 68 enzymes containing an Asp239 functional homologue have sucrose as preferential substrate while enzymes lacking this homologue use fructans as donor substrate. The presence or absence of such an Asp239 homologue is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.