The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino-acid substitution

Different plant invertase isoenzymes are characterized by a single amino-ac id difference in a conserved sequence, the WEC-P/V-D box. A proline residue is present in this sequence motif of extracellular invertase sequences, wh ereas a valine is found at the same position of vacuolar invertase sequence s. The role of this distinct difference was studied by substituting the pro line residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed mutagenesis. The mutated gene was heterol ogously expressed in an invertase-deficient Saccharomyces cerevisiae strain . The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum a nd the substrate specificity. A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extrac ellular invertases, compared to vacuolar invertases that have a valine resi due at this position.