High pressure dissociation of lactate dehydrogenase from Bacillus stearothermophilus and reconstitution of the enzyme after denaturation in 6 M guanidine hydrochloride.
Tetrameric lactate dehydrogenase from Bacillus stearothermophilus exhibits unusual stability towards high hydrostatic pressure: In contrast to the mesophilic enzyme, incubation at pressures up to 2.8 kbar does not cause irreversible denaturation. Hybridization under these conditions suggests partial dissociation to the dimer, indicating that reassociation occurs within the dead-time after pressure release (less than 20 s at less than or equal to 40 micrograms/ml, 20 degrees C). Incubation at P less than 2.8 kbar affects neither the native quaternary structure nor the catalytic function of the enzyme. Reconstitution of the unfolded and dissociated subunits after denaturation, e.g., in 6 M guanidine . HC1, is characterized by fast association favouring the native assembled structure. Evidence from spectroscopic measurements shows that reconstitution starts with a fast refolding reaction generating a native-like conformation. The subsequent rate-determining transconformation of the "structured monomers" governs the kinetics of reactivation and reassociation as one single first-order process. Chemical crosslinking with glutaraldehyde proves that the "structured monomers" undergo fast association to form the tetrameric final state of reconstitution, with significant amounts of dimeric intermediates being detectable. The renatured enzyme is indistinguishable from the native enzyme regarding its physicochemical and enzymological properties (e.g., activation by fructose-1,6-bisphosphate, and susceptibility towards proteolytic digestion).
D004355:Drug Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D011312:Pressure; D011489:Protein Denaturation; D013816:Thermodynamics
D006146:Guanidines; D007770:L-Lactate Dehydrogenase; D019791:Guanidine