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We have compared the kinetic properties (Michaelis-Menten constant [Km] and catalytic rate constant [kcat]) and amino acid sequences of orthologs of lactate dehydrogenase-A (A4-LDH) from congeners of Pacific damselfishes (genus Chromis) native to cold-temperate and tropical habitats to elucidate mechanisms of enzymatic adaptation to temperature. Specifically, we determined whether the sites of adaptive variation and the types of amino acids involved in substitutions at these sites were similar in the Chromis orthologs and other orthologs of warm-adapted and cold-adapted A4-LDH previously studied. We report striking evolutionary convergence in temperature adaptation of this protein and present further support for the hypothesis that enzyme adaptation to temperature involves subtle amino acid changes at a few sites that affect the mobility of the portions of the enzyme that are involved in rate-determining catalytic conformational changes. We tested the predicted effects of differences in sequence using site-directed mutagenesis. A single amino acid substitution in a key hinge region of the A4-LDH molecule is sufficient to change the kinetic characteristics of a temperate A4-LDH to that of a tropical ortholog. This substitution is at the same location that was identified in previous studies of adaptive variation in A4-LDH and was hypothesized to be important in adjusting Km and kcat. Our results suggest that certain sites within an enzyme, notably those that establish the energy changes associated with rate-limiting movements of protein structure during catalysis, are “hot spots” of adaptation and that common types of amino acid substitutions occur at these sites to adapt structural “flexibility” and kinetic properties. Thus, despite the wide array of options that proteins have to adjust their structural stabilities in the face of thermal stress, the adaptive changes that couple “flexibility” to alterations of function may be limited in their diversity.