Protein Surface Softness Is the Origin of Enzyme Cold-Adaptation of Trypsin

Abstract

Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution.

Enzyme surface rigidity tunes the temperature dependence of catalytic rates

“…These calculations showed that protein rigidity well outside of the active site is what seems to control the balance between thermodynamic activation parameters (Fig. 1B), and hence also the temperature adaptation of catalytic rates (9). The results showed that it is the internal energy response of the surroundings of the active site, particularly the protein-solvent interfacial surface, that differs between the cold-and warm-adapted enzymes.…”

“…However, due to the huge number of degrees of freedom involved, the extensive configurational sampling required to rigorously obtain activation enthalpies and entropies is presently beyond the scope of most quantum mechanics/molecular mechanics methods. The empirical valence bond (EVB) model (11,12) provides a very efficient method for this purpose, because it allows extensive all-atom molecular dynamics (MD) sampling of the reaction system and can be used to directly construct computational Arrhenius plots (8,9). This strategy has also recently been validated both for solution (13) and enzyme (14) reactions and was found to yield activation enthalpies and entropies in excellent agreement with experiment.…”

“…However, crystallographic analysis of psychrophilic and mesophilic trypsin has failed to identify any overall differences in flexibility between the two enzymes (7). Additionally, computer simulations of differently adapted citrate synthases and trypsins further showed that the flexibility of the active site residues is virtually identical in the psychrophilic and mesophilic enzyme variants (8,9). Moreover, structural bioinformatics analysis did not reveal any global protein descriptors that uniquely correlated with cold adaptation (3).…”

“…Recent computer simulations of cold-and warm-adapted trypsin reactions provided new clues to the problem of the origin of the enthalpy-entropy effect (9). These calculations showed that protein rigidity well outside of the active site is what seems to control the balance between thermodynamic activation parameters (Fig.…”

Enhancement of the droplet nucleation in a dense supersaturated Lennard-Jones vapor

“…Finally, Åqvist and coworkers have recently reported some very instructive studies that explored the action of coldadapted enzymes, 130 and demonstrated that the temperature adaptation is controlled by the entropic effects of residues on the protein surface, which in turn leads to entropy-enthalpy compensation (and has nothing to do with dynamical effects).…”