The rates of defined changes in protein structure during the catalytic cycle of lactate dehydrogenase.
Rapid mixing, kinetic experiments were performed on native and modified [Tyr(3NO2)237)] porcine H4 lactate dehydrogenase at low temperatures in a medium containing 30% dimethyl sulphoxide. In the temperature range -16 to +8 degrees C, the modified enzyme-NADH complex, when mixed with 1 mM pyruvate, is converted to enzyme, NAD+ and lactate at two distinctly different rates. At -16 degrees C the more rapid process occurs at a rate of 40 s-1 and the slower at 3 s-1. The slower rate is identical to that assigned to the steady-state turnover of the enzyme in these conditions and therefore reflects the slow, rate-limiting rearrangement of protein structure which has been inferred from previous kinetic experiments. The fast phase of NADH oxidation, however, proceeds at a rate which coincides with that of the closure of a loop of polypeptide over the active site of the enzyme (sensed by the nitrotyrosine group, which protonates in response to the approach of glutamate 107, a residue situated on this mobile loop). We explain these results by proposing that: (i) both the slow and fast changes in protein structure must occur before the enzyme can accomplish the redox step, (ii) the enzyme-NADH (binary) complex exists in two, slowly interconverting forms, (iii) the structural change giving rise to this slow conformational equilibrium can also occur in the ternary (enzyme-NADH-pyruvate) complex and (iv) it is this step which limits the rate of the steady-state reaction. Both of the binary forms are able to bind pyruvate, but the rate of NADH oxidation in one of the forms is rapid, since it has already undergone this slow rearrangement. In this rapidly reacting form, it is the closure of the loop (not transfer of the hydride ion) which limits the rate at which the coenzyme is oxidized, while the slowly reacting form must undergo both loop-closure and the slow structural conversion before the redox reaction can occur.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013552:Swine
D007527:Isoenzymes; D007773:Lactates; D011773:Pyruvates; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase