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Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase.

ABSTRACT: 1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme-NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pK(a) of the histidine residue in the apoenzyme is 6.8+/-0.2. In the enzyme-NADH complex it is 6.7+/-0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme-NADH complex.

SUBMITTER: Holbrook JJ

PROVIDER: S-EPMC1177532 | biostudies-other | 1973 Apr

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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Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase.

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