title: The mechanism of the bond forming events in pyridine nucleotide linked oxidoreductases. Studies with epoxide inhibitors of lactic dehydrogenase and beta-hydroxybutyrate dehydrogenase.
abstract: 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.
mesh_terms: D000818:Animals; D001665:Binding Sites; D004852:Epoxy Compounds / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors*; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013552:Swine
chemical_list: D004852:Epoxy Compounds; D004988:Ethers, Cyclic; D007770:L-Lactate Dehydrogenase; D006884:Hydroxybutyrate Dehydrogenase