Factors Affecting the Quaternary Structure of the Allosteric L‐Lactate Dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as Investigated by Hybridization and Ultracentrifugation

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Abstract
The allosteric L-lactate dehydrogenases of L. curvatus and L. casei exist in the tetrameric form (MW about 145,000) at pH 5.0-5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6-bisphosphate), but undergo reversible dissociation to monomers (MW about 35,000) at higher pH values or in the presence of urea. In the range between pH 5.5 (tetrameric state) the L. curvatus L-lactate dehydrogenase exists in a dissociation-association equilibrium comprising tetramers, dimers and monomers as indicated by the s20,wc [sedimentation coefficient] values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, and that of NADH, largely prevented dissociation. The L-lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH .gtoreq. 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH-dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the 2 dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non-allosteric ones of L. plantarum and L. acidophilus was observed.

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