Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent

Abstract

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.

Large cosolutes, small cosolutes, and dihydrofolate reductase activity

“…However, neither the relationship observed by the Mas group nor the simple theory holds. For the large enzyme YADH (150 kDa), a size dependence was observed when isopropanol was used as a substrate, but not when ethanol was used, and a size‐dependence was not observed for InhA (113 kDa) . Although the enzymes HLADH (80 kDa) and MDH (70 kDa) exhibited a size dependence, the direction of the change was opposite to that observed for LDH and alkaline phosphatase .…”

“…Notably, the Slade group showed that dextran lowers the V max of YADH for the forward (ethanol) reaction, while V max is enhanced for the reverse (isopropanol) reaction (Supporting Information, Table SII). Comparison of reactions with deuterated and nondeuterated substrates reveal that for YADH, crowding effects are linked to the rate‐determining step . Substrate‐specific crowding effects are observed for PGK, HRP, and InhA …”

“…In summary, a great deal of effort has been expended in attempts to understand how and why macromolecular crowding affects enzyme kinetics . However, a trend has not emerged, suggesting that steady‐state enzyme kinetics alone is not the best system for understanding the effects of macromolecular crowding and that more insight might be gained from combining studies on activity with studies of protein stability, folding, and diffusion.…”

“…Synthetic polymers are often used as crowding agents because they are believed to be chemically inert, although some investigators have demonstrated the existence of nonspecific protein–polymer interactions . Frequently used polymers include the branched sucrose polymer, Ficoll, the glucose polymer, dextran, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) . To distinguish between chemical effects and those arising from the macromolecular nature of the polymer, the activity in polymer solutions should be compared to activity in solutions containing monomers: sucrose, glucose, 1‐ethyl‐2‐pyrrolidone (aka N ‐ethylpyrrolidone, NEP), and ethylene glycol, respectively.…”

When both Km and Vmax are altered, Is the enzyme inhibited or activated?

“…It is also worth noting that this activation effect is fairly modest (80% increase or less in v 0 ). Such modest activation effects have been seen in the literature [6,11], and experiments are currently in progress in our laboratory to test the prediction in a statistically valid fashion.…”