Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces.
The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25 degrees C the tetramer-dimer dissociation constants for each single-mutant enzyme are similar, about 10(-6) M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramer-monomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10(-15) M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10(-28) M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.
Tolan
Dean R
DR
Biology Department, Boston University, 5 Cummington St., Boston, MA 02215, USA.
Schuler
Benjamin
B
Beernink
Peter T
PT
Jaenicke
Rainer
R
D002499:Centrifugation, Density Gradient; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D021122:Protein Subunits; D005634:Fructose-Bisphosphate Aldolase