Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase.
Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively fitted by a reversible consecutive dissociation-unfolding mechanism according to N in equilibrium 4M in equilibrium 4M (where N is the native tetramer, and M and M two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101.). At p less than or equal to 1 kbar, the pressure deactivation of both isoenzymes (H4 and M4) is described by the two-state equilibrium N in equilibrium 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M4: delta GDiss = 110 kJ/mol, delta SDiss =-860 J/K per mol, delta HDiss= -124 kJ/mol (enzyme concentration 10 microgram/ml, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be delta VDiss =-420 ml/mol (0.7 less than p less than 0.9 kbar). Measurements using 8-anilino-1-naphthalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurization beyond 1 kbar. Within the range of reversible deactivation (p less than 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. 2H2O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D005456:Fluorescent Dyes; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011312:Pressure; D013552:Swine; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007527:Isoenzymes; D046911:Macromolecular Substances; C027132:1-anilino-8-naphthalenesulfonate; D007770:L-Lactate Dehydrogenase