Hidden Conformational States and Strange Temperature Optima in Enzyme Catalysis.

The existence of temperature optima in enzyme catalysis that occur before protein melting sets in can be described by different types of kinetic models. Such optima cause distinctly curved Arrhenius plots and have, for example, been observed in several cold-adapted enzymes from psychrophilic species. The two main explanations proposed for this behavior either invoke conformational equilibria with inactive substrate-bound states or postulate differences in heat capacity between the reactant and transition states. Herein, we analyze the implications of the different types of kinetic models in terms of apparent activation enthalpies, entropies, and heat capacities, using the catalytic reaction of a cold-adapted α-amylase as a prototypic example. We show that the behavior of these thermodynamic activation parameters is fundamentally different between equilibrium and heat capacity models, and in the α-amylase case, computer simulations have shown the former model to be correct. A few other enzyme-catalyzed reactions are also discussed in this context.

Mechanistic alternatives for peptide bond formation on the ribosome.

The peptidyl transfer reaction on the large ribosomal subunit depends on the protonation state of the amine nucleophile and exhibits a large kinetic solvent isotope effect (KSIE ∼8). In contrast, the related peptidyl-tRNA hydrolysis reaction involved in termination shows a KSIE of ∼4 and a pH-rate profile indicative of base catalysis. It is, however, unclear why these reactions should proceed with different mechanisms, as the experimental data suggests. One explanation is that two competing mechanisms may be operational in the peptidyl transferase center (PTC). Herein, we explored this possibility by re-examining the previously proposed proton shuttle mechanism and testing the feasibility of general base catalysis also for peptide bond formation. We employed a large cluster model of the active site and different reaction mechanisms were evaluated by density functional theory calculations. In these calculations, the proton shuttle and general base mechanisms both yield activation energies comparable to the experimental values. However, only the proton shuttle mechanism is found to be consistent with the experimentally observed pH-rate profile and the KSIE. This suggests that the PTC promotes the proton shuttle mechanism for peptide bond formation, while prohibiting general base catalysis, although the detailed mechanism by which general base catalysis is excluded remains unclear.

Catalytic Adaptation of Psychrophilic Elastase.

The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme, and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures has actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates, the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined, and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is caused by redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface-exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.

Computational design of the temperature optimum of an enzyme reaction.

Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog.

Computer simulations explain the anomalous temperature optimum in a cold-adapted enzyme.

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.

Towards Rational Computational Engineering of Psychrophilic Enzymes.

Cold-adapted enzymes from psychrophilic species achieve their high catalytic efficiency at low temperature by a different partitioning of the activation free energy into its enthalpic and entropic components, compared to orthologous mesophilic enzymes. Their lower activation enthalpy, partly compensated by an increased entropic penalty, has been suggested to originate from changes in flexibility of the protein surface. Multiple sequence alignments of psychrophilic and mesophilic enzymes also show characteristic motifs located in surface loops of the protein. Here, we use computer simulations to examine the effects of a number of designed surface mutations of psychrophilic and mesophilic elastases on the temperature dependence of the catalyzed peptide cleavage reaction. For each of 14 mutant enzyme variants we report calculations of their thermodynamic activation parameters. The results show that substitution of psychrophilic loop residues into the mesophilic enzyme consistently changes both the activation parameters and loop flexibilities towards the former, and vice versa for opposite substitutions.