|Title||Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed.|
|Publication Type||Journal Article|
|Year of Publication||2017|
|Authors||Carlin DAlexander, Hapig-Ward S, Chan BWayne, Damrau N, Riley M, Caster RW, Bethards B, Siegel JB|
|Keywords||Algorithms, Catalytic Domain, Cloning, Molecular, Crystallography, X-Ray, Enzyme Stability, Escherichia coli, Genetic Variation, Glycoside Hydrolases, Kinetics, Models, Molecular, Protein Structure, Tertiary, Temperature|
Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.
|Alternate Journal||PLoS ONE|
|PubMed Central ID||PMC5439667|