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Slow structural changes shown by the 3-nitrotyrosine-237 residue in pig heart [Tyr(3NO2)237] lactate dehydrogenase.

ABSTRACT: 1. The pKa of the phenolic hydroxy group of the Tyr(3NO2)-237 residue in pig heart [Tyr(3NO2)237]lactate dehydrogenase is 7.2 in the apoenzyme, 7.4 in the enzyme-NADH complex and 7.8 in the enzyme-NADH-oxamate complex. The alkaline shift from apoenzyme to ternary complex is ascribed to the approach of the Glu-107 residue during the movement of the polypeptide loop residues 98-110. 2. The affinities of the nitrated enzyme for NADH and for oxamate (in the presence of NADH) are slightly less than those of the native enzyme. The turnover number for the nitrated enzyme in the pyruvate-to-lactate direction is about 0.75 of the value for the native enzyme. 3. Temperature-jump relaxation experiments of the enzyme saturated with NADH but fractionally saturated with oxamate are interpreted to show that the pKa of the nitrotyrosine residue responds to a protein rearrangement after oxamate binds to the binary enzyme-NADH complex. 4. Transient-kinetic experiments show the environment of the Tyr(3NO2)-237 residue in the enzyme-NADH-pyruvate complex of the steady state to be similar to that in the enzyme-NADH-oxamate inhibitor complex.

SUBMITTER: Parker DM

PROVIDER: S-EPMC1163670 | biostudies-other | 1982 Mar

REPOSITORIES: biostudies-other

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Project description:Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

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Slow structural changes shown by the 3-nitrotyrosine-237 residue in pig heart [Tyr(3NO2)237] lactate dehydrogenase.

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