Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase
| Autor: | Kristin M. Slade, Xander E Wilcox, Charmaine B. Chung |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Michaelis-menten dex dextran (number afterward represents the molecular weight of the polymer in kDa) QH301-705.5 Vmax maximal rate under Michaelis-Menten kinetics Biophysics Ficoll PVP polyvinylpyrrolidone QD415-436 Michaelis–Menten kinetics Biochemistry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Km is Michaelis constant Biology (General) Dextran Alcohol dehydrogenase Depletion layer PEG polyethylene glycol biology Steady state kinetics Acetaldehyde KD dissociation constant Dissociation constant 030104 developmental biology chemistry 030220 oncology & carcinogenesis kDa kilodaltons Excluded volume biology.protein Macromolecular crowding Research Article |
| Zdroj: | Biochemistry and Biophysics Reports Biochemistry and Biophysics Reports, Vol 26, Iss, Pp 100956-(2021) |
| ISSN: | 2405-5808 |
| Popis: | 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 Vmax and Km for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in Vmax 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. Highlights • Yeast alcohol dehydrogenase reduction of acetaldehyde is enhanced by crowding. • Crowding effects on YADH kinetics depend on the direction of the reaction. • Crowders like dextran can be used as a tool to elucidate enzyme mechanism. • Excluded volume optimizes YADH hydride transfer; viscosity hinders product release. • The presence of a depletion layer with large crowders mitigates their effects. |
| Databáze: | OpenAIRE |
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