The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Abstract

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.

Pressure relaxation of the equilibrium of the pig heart lactate dehydrogenase system

“…Many of the rate constants in Table 3 were taken from the results of steady-state and transient kinetic experiments. The value ofk+4 given by Whitaker et al (1974) is 1200s-1; however, allowing for the contribution of the steady-state rate to the burst rate constant (Gutfreund, 1975, p. 200) decreases k+4 to about 9(ls-1. The rate-limiting step for single-turnover pyruvate reduction at low pH is taken to be k-4, and the observed pH-dependence (Boland & Gutfreund, 1975) suggests that k-4 is about 800s-1.…”

“…The rate-limiting step for single-turnover pyruvate reduction at low pH is taken to be k-4, and the observed pH-dependence (Boland & Gutfreund, 1975) suggests that k-4 is about 800s-1. The rate constants k+3 and k-3 are unknown, but their ratio is defined by the proportion of hidden transient (phase 1) in lactate oxidation (0.3 at pH 8.0; Whitaker et al, 1974). Both k+3 and k3 are taken to be faster than 10OOs-', since k+3, the slower one, controls the hidden phase in the stopped-flow experiments (Whitaker et al, 1974).…”

“…As a test of the mechanism, computer simulations were used to compare Germany (batch 1046318). Stock solutions were prepared by desalting on a column of Sephadex G-25 and protein concentrations were determined as described by Whitaker et al (1974). The active-site concentration of enzyme solutions, determined by titration with NADH, was 80% of the protein concentration as described by Whitaker et al (1974).…”

“…The value of k+, must be much greater than k-4 to give the full bursts observed in lactate oxidation (Whitaker et al, 1974). Also, the second-order rate constant for pyruvate binding, k7, must be set at a value that is not unreasonably fast.…”

“…Transient kinetic analysis of stopped-flow experiments in this laboratory (Whitaker et al, 1974; Boland & Gutfreund, 1975;Holbrook et al, 1976) has resulted in considerable elucidation of our understanding of the reaction of lactate dehydrogenase with its substrates and allows pressure-relaxation effects to be analysed in * Present address:…”