Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus.
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)
Kotik
M
M
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Zuber
H
H
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003903:Deuterium; D005454:Fluorescence Polarization; D005635:Fructosediphosphates / Q000494:pharmacology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D005635:Fructosediphosphates; D046911:Macromolecular Substances; D009243:NAD; D003903:Deuterium; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate