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

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• 作者：Samuel H. Schneider ; Schuyler P. Lockwood ; Dominique I. Hargreaves ; David J. Slade ; Micaela A. LoConte ; Bridget E. Logan ; Erin E. McLaughlin ; Michael J. Conroy ; Kristin M. Slade
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：September 29, 2015
• 年：2015
• 卷：54
• 期：38
• 页码：5898-5906
• 全文大小：403K
• ISSN：1520-4995

文摘

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and 鈥渋nert鈥? 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鈥揗enten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased V_max values directly correlate with slower translational diffusion and the decreased K_m values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer鈥檚 ability to decrease K_m is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in V_max values for glucose 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.