Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄ orthologs of Antarctic notothenioid fishes

Abstract

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A₄ (A₄-LDH) from nine Antarctic (−1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis–Menten constants (K _m) and catalytic rate constants (k _cat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in k _cat and K _m are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A₄-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting k _cat, we conclude that notothenioid A₄-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in K _m and k _cat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase k _cat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high K _m values relative to warm-adapted orthologs.