Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus

Abstract

L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C. To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange. In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP. The results obtained from these measurements at various temperatures can be summarized as follows. No changes in the secondary-structure distribution are detectable from far-UV CD measurements. On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur. With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C. Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature. It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme. We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C. The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

“…3). In other cases, even marginal structural effects have been detected upon temperature variation (Kotik and Zuber, 1992). In the present case, the slight decrease in asymmetry in the environment of the aromatic chromophores may indicate the increase in tlexibility of the polypeptide chain at elevated temperature.…”

Extremely thermostable L(+)‐lactate dehydrogenase from thermotoga maritima: Cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state

“…Tetramers and octamers can be separated as homogeneous entities undergoing reassociation after preceding complete dissociation and denaturation, e.g., in 6 M guanidinium chloride. With these characteristics they differ from aggregates of BsLDH observed under pre-denaturation conditions at 55 "C (Kotik & Zuber, 1992). Similarly, they cannot be related to the concentration-dependent self-association of bovine liver glutamate dehydrogenase (Eisenberg et al, 1976).…”

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

“…The red shift of emission could be attributed to an increase in the mean environment polarity of the Nile Red binding site, as the steady state intensity was reduced and lifetime was decreased ( 121 ). Other examples are the application of Nile Red fluorescence for probing protein surface hydrophobicity of L-lactate dehydrogenase from Bacillus stearothermophilus ( 122 ) and photoactive yellow protein from Halorhodospira halophila ( 123 ).…”