Disruption of the aldolase A tetramer into catalytically active monomers.
The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.
Beernink
P T
PT
Biology Department, Boston University, MA 02215, USA.
Tolan
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003196:Computer Graphics; D018360:Crystallography, X-Ray; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014508:Urea
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014508:Urea; D005634:Fructose-Bisphosphate Aldolase