A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure.
We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.
D000494:Allosteric Regulation; D000495:Allosteric Site; D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005635:Fructosediphosphates / Q000494:pharmacology; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D001426:Bacterial Proteins; D005635:Fructosediphosphates; D011773:Pyruvates; D005973:Glutamine; D009243:NAD; D019289:Pyruvic Acid; D001120:Arginine; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate