Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on XXXX Dehydrogenase Steady-State Kinetics.

@entity9588 @entity375 @entity285 @entity413
UNASSIGNED: To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of @entity375 @entity285 dehydrogenase (YADH) than its small molecule counterpart, @entity413 . For @entity413 , the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as @entity413 , this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that @entity413 and dextran have surprisingly similar @entity9588 diffusion coefficients. Thus, the reduction in Vmax values for @entity413 is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.
