The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively

Oligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of beta-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of beta-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of L-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits.

Automated summary

Oligomeric enzymes are known for their higher catalytic rates compared to monomeric enzymes, but the extent of additivity in their activity is still not well understood. This study used tetrameric rabbit lactate dehydrogenase as a model to examine the kinetics of its catalytic action. Surprisingly, when the concentration of the limiting reactant exceeded that of a single subunit, there was a significant slowdown in the enzyme's conformational rearrangements.