Studies on Rates of Abortive Ternary Complex Formation of Lactate Dehydrogenase Isozymes

Rates of formation and dissociation of an abortive ternary complex between lactate dehydrogenase isozymes from various mammalian tissues, NADf, and pyruvate were studied utilizing stopped-flow spectrofluorometry.Formation of the complex was accompanied by a decrease in protein fluorescence at 340 rnp and a loss of lactate dehydrogenase activity.Rates of formation of an abortive lactate dehydrogenase-1 and lactate dehydrogenase-5 complex were compared at 25' and 40' over a range of pyruvate and enzyme concentrations, at two NAD+ concentrations, and with two coenzyme analogues.Dialysis against buffer produced an increase in protein fluorescence and recovery of lactate dehydrogenase activity that suggested dissociation of the abortive complex.At pyruvate concentrations of 0.3 mu the abortive ternary complex formed twice as fast with lactate dehydrogenase-1 as with lactate dehydrogenased and with 40.0 mu pyruvate it formed 5 times faster.The rate of complex formation increased with both isozymes when pyruvate concentrations were raised from 0.3 mu to 40.0 mM and when the temperature was elevated from 25" to 40'.However, the rates of abortive complex formation were inversely related to enzyme concentration.Compared to lactate dehydrogenase turnover numbers, the rates of formation of the abortive ternary complex were slow.Under physiological conditions of temperature (40") and pyruvate concentration (0.3 to 1.0 maa), half the maximum fluorescence quenching required approximately 50 set for partially purified lactate dehydrogenase-1.Changes in coenzyme concentration from 0.10 rnrd to 0.75 mu failed to alter the rates of abortive complex formation.With the 3-acetylpyridine analogue of NAD+, rates of abortive complex formation for both lactate dehydrogenase-1 and lactate dehydrogenase-5 were similar, whereas with deamino-NAD+ the abortive complex formed faster with lactate dehydrogenase-1 than with lactate dehydrogenase-5.Dialysis of the abortive complex resulted in recovery of enzyme activity; the rate of recovery was faster with lactate dehydrogenase-5 than with lactate dehydrogenase-1.