The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase.
1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3mum and from the muscle isoenzyme 25mum at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme-NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme-NADH-substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD(+). The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.
D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D006038:Glyoxylates; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007536:Isomerism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D009574:Nitro Compounds; D010667:Phenylpyruvic Acids; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013552:Swine; D013997:Time Factors
D000596:Amino Acids; D006038:Glyoxylates; D007527:Isoenzymes; D009574:Nitro Compounds; D010667:Phenylpyruvic Acids; D009243:NAD; D006639:Histidine; D007770:L-Lactate Dehydrogenase