Structural determinants of cold adaptation and stability in a large protein

The heat-labile alpha -amylase from an antarctic bacterium is the largest k nown protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry, Mutants of this enzyme were pr oduced, carrying additional weak interactions found in thermostable alpha - amylases, It is shown that single amino acid side chain substitutions can s ignificantly modify the melting point T-m, the calorimetric enthalpy DeltaH (cal) the cooperativity and reversibility of unfolding, the thermal inactiv ation rate constant, and the kinetic parameters k(cat) and K-m. The correla tion between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches o r aggregations typical of large proteins. Although all mutations were locat ed far from the active site, their overall trend is to decrease both k(cat) and K-m by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapte d alpha -amylase has lost a large number of weak interactions during evolut ion to reach the required conformational plasticity for catalysis at low te mperatures, thereby producing an enzyme close to the lowest stability allow ing maintenance of the native conformation.