The mechanism of the bond forming events in pyridine nucleotide linked oxidoreductases. Studies with epoxide inhibitors of lactic dehydrogenase and beta-hydroxybutyrate dehydrogenase.

2,3-Epoxybutyrate and 2,3-epoxypropionate act as effective competitive inhibitors of pig heart lactic dehydrogenase. KIapp for both inhibitors was pH dependent and varied according to the general equation KIapp = KI(1 +Ka/H+) which may be predicted if the binding of the epoxide to the E-NADH complex involves a compulsory protonation step. Values of KI(epoxybutyrate), KI(epoxypropionate) and pKa were estimated as 150 muM, 860 muM, and 6.8, respectively. The formation of an E-NADH epoxide inhibitor complex was followed directly by fluorescence measurements. Both epoxybutyrate and epoxypropionate enhanced fluorescence of the E-NADH complex and caused a 20-nm blue shift in the maximum emission wavelenght. The dissociation constants measured by fluorescence titration for both epoxides increased as the pH was raised reflecting a decreased affinity for the E-NADH complex. 2,3-Epoxybutyrate was also shown to inhibit beta-hydroxybutyrate dehydrogenase by a mechanism which is consistent with compulsory protonation prior to addition of the epoxide. These results are discussed in terms of a general mechanism for the bond forming events in pyridine nucleotide linked oxidore-ductases.     

Pressure-induced structural changes of pig heart lactic dehydrogenase

Lactic dehydrogenase from pig heart can be reversibly dissociated at hydrostatic pressures above 1000 bar. The breakdown of the native quaternary structure occurs at lower pressures compared to the isoenzyme from pig, skeletal muscle. As shown by hybridization experiments of the two isoenzymes the final product of dissociation is the homogeneous monomer. Fluorescence emission spectra of the monomeric enzyme at elevated pressure are characterized by a decrease in fluorescence intensity without any red shift, indicating that no significant unfolding occurs upon high-pressure dissociation. The spectral changes are comparable to those observed after acid dissociation. The amount and rate of deactivation depend on pressure and on the conditions of the solvent. The presence of various anions (Cl^?, SO^2?₄. HPO₄^2?) has no effect on the stability of ihe enzyme towards pressure. High-pressure denaturation (as monitored by intrinsic protein fluorescence), and deactivalion (measured immediately after decompression) run parallel; the pressure dependence of their first-order rate constants is characterized by an activation volume ΔV^≠_Dc = ?140 = 10 cm³/mol. As taken from the yield of reconstitution, dissociation, denaturation and deactivation are found to be fully reversible provided the pressure does not exceed a limiting value (p = 1000 bar in Tris. pH 7.6: 24 h incubation at 20°C). After extended incubation beyond the limiting, pressure of 1000 bar. “irreversible high-pressure denaturation” occurs which is accompanied bv partial aggregation after decompression. The coenzyme, NAD⁺ stabilizes the native tetramer shifting the dissociation equilibrium to higher pressures. The overall dissociation-association reaction can be quantitatively described by a consecutive dissociation/unfolding mechanism N?4 M^'?4 M^☆ (where N is the native tetramer. and M^' and M^☆ two different conformations of the monomer). The reaction volume of the dissociation reaction N?4 M^' is found to be ΔV_Diss = ?360 = 30 cm³/mol: as indicated by the pressure dependence of the yield of reconstitution, the reaction volume of the equilibrium M^'?M^XXX is also negative.