Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

Abstract

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V max and K m for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in V max is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD + product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this “tuning” of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.

Tunable In Vivo Co-localisation of Enzymes Within P22 Capsid-Based Nanoreactors

“…This is consistent with previous findings for other enzymes individually encapsulated within VLPs that have shown high loading densities can lead to reduced activity (Fiedler et al, 2010;Minten et al, 2011). The decrease in enzyme activity as a result of apparent molecular crowding may be attributed to factors such as conformational changes and excluded volume effects (Norris and Malys, 2011;Wilcox et al, 2021). On the other hand, for alcohol dehydrogenase (AdhD) encapsulated at different densities in P22 VLPs, an increase in enzymatic activity was observed with an increase in AdhD loading (Sharma and Douglas, 2020).…”

Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors

“…6,14 The decrease in enzyme activity as a result of apparent molecular crowding may be attributed to factors such as conformational changes and excluded volume effects. 42,43 On the other hand, for alcohol dehydrogenase (AdhD) encapsulated at different densities in P22 VLPs, an increase in enzymatic activity was observed with an increase in AdhD loading. 5 However, wild-type SP induced macromolecular crowding in a concentration-dependent manner when coencapsulated with AdhD, suggesting that AdhD did not exhibit self-crowding but is affected by crowding in the presence of other proteins.…”

Parameter Reliability and Understanding Enzyme Function

“…As might be expected, cellular crowding affects diffusion rates, as well as protein aggregation and phase separation [ 74 ]. Many attempts to quantify these effects have used ‘inert’ crowding additives, such as dextran, ficol, or polythylene glycol [ 75 , 76 ]. Although such studies indicated the kinetic behaviour to be selectively affected, the nature of the crowding agent itself was found to be an important factor [ 74 , 77 ].…”