Abstract
Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic α-amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 °C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima.
Highlights
Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs
It is generally accepted that the main adaptive feature of psychrophilic enzymes is the redistribution of the thermodynamic activation parameters compared with mesophilic and thermophilic orthologs, while the activation free energies are usually similar at room temperature[1,2,3,4,5]
This means that the evolutionary pressure on protein stability at the physiological working temperature is bound to be considerably weaker for psychrophilic enzymes, which may be one reason for why their Tm has drifted toward lower values compared with mesophilic orthologs[5,6]
Summary
Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Computer simulations that directly evaluated free energy barriers for the catalyzed reactions[9,10,11,12,13] have shown that the mobility of active-site residues is generally very similar in psychrophilic and mesophilic enzyme orthologs, while surface loop mobilities may differ considerably and strongly affect the balance between ΔH‡ and ΔS‡ This finding is entirely in line with the fact that multiple sequence alignments between orthologous psychrophilic and mesophilic enzymes reveal that characteristic mutations are typically located in such loop regions[9,10,11]
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