More than three-quarters of the Earth's surface is cold — oceans with a constant temperature of 4–5°C below a depth of 1,000m cover approximately 70% of the Earth's surface. The microorganisms that occupy these regions are known as psychrophiles. To maintain essential chemical reactions at these temperatures, psychrophilic enzymes are cold active and heat labile. Psychrophilic enzymes maintain high activity at low temperatures mainly by decreasing the temperature dependence of the reaction that is catalysed. This is achieved by improving the mobility or flexibility of the active site. As a consequence, substrate binding is generally less efficient, but specific mutations can compensate for this adaptive drift, especially when substrate binding (Km) has a regulatory function. The catalytic centre of cold-active enzymes is identical to that of mesophilic enzymes, to maintain specificity, but local interactions might help to improve catalysis at low temperatures, such as better accessibility to the active site or favourable electrostatic interactions with the substrate. Generally, adaptive mutations favouring active-site flexibility are located outside the catalytic centre. All known interaction types that stabilize a protein are reduced in number and strength, but each enzyme family uses one or a combination of the altered interactions to gain in molecular mobility. At least in the case of the best-studied psychrophilic enzyme (chitobiase), the relationships between stability and activity at low temperatures have been shown by site-directed mutagenesis. Stabilizing the psychrophilic enzyme, by engineering the weak interactions found in the mesophilic enzyme, decreases activity and improves substrate binding of the mutants. The stability curves of psychrophilic enzymes reveal several unsuspected properties. They are optimally stable at room temperature, which reflects the dominant effect of hydrophobic forces in protein folding. However, they are cold labile and more prone to cold denaturation than mesophilic proteins, which is a phenomenon that might set a biophysical lower limit to life at low temperatures. In addition, the thermodynamic contributions to their stability are the opposite to that of mesophilic proteins, for example the stability of cold-active enzymes is entropy-driven at low temperatures. Directed evolution experiments show that several molecular adjustments can lead to cold activity. However, in cold environments, the simplest strategy seems to be to lose stability, in the absence of selection for stable proteins, to gain in flexibility and activity, under a strong selective pressure for cold-active enzymes.
