Identification of amino acids essential for enzymatic activity of plant invertases

The role of two conserved sequence motifs in plant invertases, the NDPN and WEC-P/V-D boxes, was studied by a structure-function analysis of extracell ular invertase CIN1 from Chenopodium rubrum. Specific amino acid substituti ons were introduced by site-directed mutagenesis, and the mutated genes wer e heterologously expressed in an invertase deficient Saccharomyces cerevisi ae strain. It was shown that the aspartate from the first and the glutamate and the cysteine from the second box are essential for enzyme activity. Th e possible function of these amino acid residues is discussed with respect to the inhibitory effect of sulfhydryl group blocking reagents on invertase activity, the interaction between amino acids in the active centre, and th eir participation in the catalytic mechanism.