EVOLUTION OF LACTATE DEHYDROGENASE-A HOMOLOGS OF BARRACUDA FISHES (GENUS SPHYRAENA) FROM DIFFERENT THERMAL ENVIRONMENTS - DIFFERENCES IN KINETIC-PROPERTIES AND THERMAL-STABILITY ARE DUE TO AMINO-ACID SUBSTITUTIONS OUTSIDE THE ACTIVE-SITE

Orthologous homologs of lactate dehydrogenase-a (LDH-A) (EC 1.1.1.27; NAD(+):lactate oxidoreductase) of six barracuda species (genus Sphyrae na) display differences in Michaelis-Menten constants (apparent K-m) f or substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in K-m with incr easing measurement temperature occur for all homologs, yet K-m at norm al body temperatures is similar among species because of the inverse r elationship between adaptation temperature and K-m. Thermal stabilitie s of the homologs also differ. To determine the amino acid substitutio ns responsible for differences in K-m and thermal stability, peptide m apping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar pe ptide maps, those of the north temperate species, S. argentea, the sub tropical species, S. lucasana, and the south temperate species, S. idi astes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzyme s. The sequence of LDH-A from S. lucasana differs from that of S. idia stes only at position 8. The homolog of S. argentea differs from the o ther two sequences at positions 8, 61, 68, and 223. We used a full-len gth cDNA clone of LDH-A of S. lucasana to test, by site-directed mutag enesis, the importance of these sequence changes in establishing the o bserved differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences i n K-m between the LDH-As of S. argentea and S. lucasana. Differences a t position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary ada ptation of proteins to temperature thus may be achieved by minor chang es in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.