The molecular pathway by which fructose 1,6-bisphosphate induces the assembly of a bacterial lactate dehydrogenase

Abstract

Lactate dehydrogenase from Bacillus stearothermophilus exists in two interconverting states of subunit assembly, the dimer (d) and the tetramer (t). Fructose 1,6-bisphosphate (Fru-1,6- P 2) can bind to either of these forms of the enzyme: tightly to the tetramer and weakly to the dimer. The binding of this ligand results in an increase in the affinity for pyruvate and a stabilization of the tetrameric form of the enzyme. We have elucidated the routes of assembly and disassembly of the protein in response to changed levels of this phosphorylated sugar at pH 6 and at 25°C and our findings are the following (1) the dimeric form of the enzyme binds a single Fru-1,6- P 2) (f) molecule (to form d-f) in a rapid equilibrium event with a dissociation constant of about 3 mM. (2) Two of these d-f intermediates are able to combine to form the liganded tetramer (t-f 2) at a rate which is limited by their rate of diffusion through the medium i.e., no slow structural rearrangement of the subunit interfaces limits the rate of formation of this tetramer. The dimer-dimer interface in the t-f 2 species is extremely stable, having a K d of 3·10 −10 M (and a rate of dissociation of 8 · 10 −5 s −1). (4) If the Fru-1,6- P 2-stabilized tetramer (t-f 2) is forced to dissociate by rapid and extreme dilution, the protein first loses its ligands (at a rate of about 10 −2 s −1) and the dimer-dimer interface is then sufficiently destabilized to dissociate rapidly (greater than 1 s −1 yielding unliganded dimers. (5) The tetramer can accommodate only two Fru-1,6- P 2 molecules and the binding of this activator at one site has only a small effect upon binding at the remaining site. The rate at which Fru-1,6- P 2 can bind to and stabilize the tetramer (less than 10 3 M −1 · s −1) is much slower than expected for a diffusion-controlled process. These kinetic observations are consistent with the existence in the tetramer of two activator binding sites which are buried on subunit interfaces. These sites are in a central hole within the tetramer and ‘cage’ the Fru-1,6- P 2 molecules. We attribute the stability of the liganded tetramer to the diphosphorylated sugar molecule being able to bridge and thus stabilize an otherwise weak protein-protein interface, with one of its phosphate groups bound to each contributing dimer. In the dimeric form of the enzyme the subunit interface which carries the activator site is open to the solvent and Fru-1,6- P 2 binding is rapid but weak.