An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P-2] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in the absence of Fru(1,6)P-2 under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P-2-binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P-2 or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P-2 under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P-2-independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P-2 requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P-2, through a slight decrease and increase of the Fru(1,6)P-2 sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P-2. The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.