JOHNS, G.C.*; SOMERO, G.N.: Evolution of A₄-lactate dehydrogenase in warm-stenothermal environments: investigating biochemical adaptation in damselfishes (Pomacentridae).

Building on previous work elucidating the nature of biochemical adaptation of muscle-type lactate dehydrogenase (A₄-LDH) in cold-stenothermal Antarctic Notothenioids and eurythermal gobies, we investigated functional differences associated with evolution in a warm-stenothermal environment. To address this, we analyzed genetic and biochemical properties of A₄-LDH for several species of damselfishes. The true clownfish (Amphiprion percula) has a highly restricted distribution in the Tropical Western Pacific where temperatures average 28.5°C, while the Garibaldi (Hypsypops rubicundus) from the NE Pacific experiences 10-22°C. Kinetic analyses of purified A₄-LDH show a lower Michaelis constant (K_m) across 5-35°C for the clownfish relative to the Garibaldi, such that the two species show comparable substrate binding efficiencies at each species� respective physiological temperatures. Interestingly, while observed K_m trends agree well with predictions from previous studies, a concomitantly lower catalytic constant (k_cat) for the clownfish � as would be predicted from earlier studies � was not detected. Sequence analysis of the predicted amino acid sequence from multiple independent clones of ldh-a cDNA for each species reveals three substitutions between species. Two of these occur in the exact locations already demonstrated to be of critical importance in cold-stenothermy adaptation. Together, these data reveal an intriguing comparison and contrast to previous studies of A₄-LDH, reinforcing the contention that changes in certain key positions away from the catalytic site can cause large functional differences in enzyme kinetics.