Crowding-Induced Uncompetitive Inhibition of Lactate Dehydrogenase: Role of Entropic Pushing.

Abstract

The cell is an extremely complex environment, notably highly crowded, segmented, and confining. Overall, there is overwhelming and ever-growing evidence that to understand how biochemical reactions proceed in vivo, one cannot separate the biochemical actors from their environment. Effects such as excluded volume, obstructed diffusion, weak nonspecific interactions, and fluctuations all team up to steer biochemical reactions often very far from what is observed in ideal conditions. In this paper, we use Ficoll PM70 and PEG 6000 to build an artificial crowded milieu of controlled composition and density in order to assess how such environments influence the biocatalytic activity of lactate dehydrogenase (LDH). Our measurements show that the normalized apparent affinity and maximum velocity decrease in the same fashion, a behavior reminiscent of uncompetitive inhibition, with PEG resulting in the largest reduction. In line with previous studies on other enzymes of the same family, and in agreement with the known role of a surface loop involved in enzyme isomerization and regulation of access to the active site, we suggest that the crowding matrix interferes with the conformational ensemble of the enzyme. This likely results in both impaired enzyme-complex isomerization and thwarted product release. Molecular dynamics simulations confirm that excluded-volume effects lead to an entropic force that effectively tends to push the loop closed, thereby effectively shifting the conformational ensemble of the enzyme in favor of a more stable complex isoform. Overall, our study substantiates the idea that most biochemical kinetics cannot be fully explained without including the subtle action of the environment where they take place naturally, in particular accounting for important factors such as excluded-volume effects and also weak nonspecific interactions when present, confinement, and fluctuations.