The lactate dehydrogenase–reduced nicotinamide–adenine dinucleotide–pyruvate complex. Kinetics of pyruvate binding and quenching of coenzyme fluorescence

The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3°C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex ENADHPyr. Even though the oxidation–reduction reaction rapidly follows the formation of this complex, the numerical values of k−4 (8.33×105m−1·s−1) and k+4 (222s−1) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation–reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of ENAD+Lac in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a `burst reaction' in which ENAD+Lac is produced, and this estimation yields, from kobs.=120s−1, k−2=1200s−1.