To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A 4 (A 4 -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 A 4 -LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting k cat , we conclude that notothenioid A 4 -LDHs have adapted to cold temperatures by increases in f lexibility 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 temperatureadaptive variation in K m and k cat . Changes in sequence that increase f lexibility 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 warmadapted orthologs.

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Temperature profoundly influences physiological responses in animals, primarily due to the effects on biochemical reaction rates. Since physiological responses are often exemplified by their rate dependency (e.g., rate of blood flow, rate of metabolism, rate of heat production, and rate of ion pumping), the study of temperature adaptations has a long history in comparative and evolutionary physiology. Animals may either defend a fairly constant temperature by recruiting biochemical mechanisms of heat production and utilizing physiological responses geared toward modifying heat loss and heat gain from the environment, or utilize biochemical modifications to allow for physiological adjustments to temperature. Biochemical adaptations to temperature involve alterations in protein structure that compromise the effects of increased temperatures on improving catalytic enzyme function with the detrimental influences of higher temperature on protein stability. Temperature has acted to shape the responses of animal proteins in manners that generally preserve turnover rates at animals' normal, or optimal, body temperatures. Physiological responses to cold and warmth differ depending on whether animals maintain elevated body temperatures (endothermic) or exhibit minimal internal heat production (ectothermic). In both cases, however, these mechanisms involve regulated neural and hormonal over heat flow to the body or heat flow within the body. Examples of biochemical responses to temperature in endotherms involve metabolic uncoupling mechanisms that decrease metabolic efficiency with the outcome of producing heat, whereas ectothermic adaptations to temperature are best exemplified by the numerous mechanisms that allow for the tolerance or avoidance of ice crystal formation at temperatures below 0°C. © 2012 American Physiological Society. Compr Physiol 2:2151‐2202, 2012.