Abstract
The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.
Highlights
Cold-adapted enzymes remain catalytically active at low temperatures
The mutants Mut5 and Mut5CC from a psychrophilic ␣-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic ␣-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. These mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes
We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding
Summary
Results: Mutants of a cold-adapted ␣-amylase stabilized by engineered weak interactions and a disulfide bond have lost the kinetic optimization to low temperatures. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding To this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity. These multiple mutants provide clear insights into the structure-function relationships allowing psychrophilic enzymes to fulfill their biochemical functions at low temperatures
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