title: 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-bisphosphate and activate the enzyme only if intact, (ii) hybrids with only one functional fructose 1, 6-bisphosphate-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.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Fushinobu
authors.forename: K
authors.initials: K
authors.lastname: Kamata
authors.forename: S
authors.initials: S
authors.lastname: Iwata
authors.forename: H
authors.initials: H
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Ohta
authors.forename: H
authors.initials: H
authors.lastname: Matsuzawa
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology*; D001665:Binding Sites; D004926:Escherichia coli; D005635:Fructosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D005635:Fructosediphosphates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate