Allosteric activation of L-lactate dehydrogenase analyzed by hybrid enzymes with effector-sensitive and -insensitive subunits

Abstract

Subunit-hybrid enzymes of mutant tetrameric L-lactate dehydrogenases from Bifidobacterium longum were studied in an examination of the mechanism of allosteric activation by fructose 1,6-bisphosphate. We earlier developed an in vivo method for subunit hybridization in Escherichia coli and the hybrids formed were a mixture with different subunit compositions. The B. longum hybrids were separated by anion-exchange chromatography with a mutational tag. Hybrids formed between fructose 1,6-bisphosphate- desensitized subunits and wild-type subunits and also between fructose 1,6- bisphosphate-desensitized subunits and catalytically inactive subunits. Kinetic analyses of the hybrid enzymes showed that (i) those residues from two symmetrically related subunits that constituted the fructose 1,6- bisphosphate-binding site could bind fructose 1,6-hisphosphate and activate the enzyme only if intact, (ii) hybrids with only one functional fructose 1,6-hisphosphate-binding site were fully sensitive to fructose 1,6- bisphosphate, but the allosteric equilibrium had shifted partially, and (iii) activation by fructose 1,6-bisphosphate at the fructose 1,6-bisphosphate- binding site was transmitted to the active sites through a quaternary structural change, not through direct conformational change within a subunit. These results are evidence of the validity of the concerted allosteric model of this enzyme based on T- and R-state structures in the same crystal lattice proposed earlier.