Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent.
Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand the ramifications of this crowding, the Michaelis-Menten kinetics of yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased the maximal rate of the reaction by 20-40%. This work reveals the importance of considering enzyme mechanism when evaluating the ways in which crowding can alter kinetics.
Department of Chemistry, Hobart and William Smith Colleges , Geneva, New York 14456, United States.
A E
AE
Wilcox
Department of Chemistry, Hobart and William Smith Colleges , Geneva, New York 14456, United States.
Micaela A
MA
LoConte
Department of Chemistry, Hobart and William Smith Colleges , Geneva, New York 14456, United States.
Kristin M
KM
Slade
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism*; D004058:Diffusion; D000431:Ethanol / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014783:Viscosity
D003911:Dextrans; D046911:Macromolecular Substances; D009243:NAD; D000431:Ethanol; D000426:Alcohol Dehydrogenase