PubMed1997
We compared the deduced amino acid sequences, heat stabilities and thermal sensitivities of a kinetic property, the apparent Michaelis­Menten constant (Km) of pyruvate, of A4-lactate dehydrogenase (A4-LDH) in four species of goby fishes (Family Gobiidae), adapted to different temperatures, to examine how changes in primary structure influence the adaptation of enzymes. The effect of temperature on Km of pyruvate reflected each species' environmental temperature. For the most eurythermal species, Gillichthys seta, which is endemic to shallow intertidal regions of the upper Gulf of California and encounters temperatures between approximately 9 and 40 °C, Km of pyruvate was minimally affected by temperature, compared with the A4-LDH orthologues from a less eurythermal congener, G. mirabilis (9­30 °C), a cold temperate goby, Coryphopterus nicholsi (10­18 °C) and a tropical species, C. personatus (25­32 °C). Heat denaturation profiles failed to correlate with habitat temperature; G. mirabilis A4-LDH was most thermally stable, followed by the orthologues of C. nicholsi and G. seta. Complementary DNAs (cDNAs) encoding LDH-As of G. seta, Gulf of California and Pacific coast populations of G. mirabilis and C. nicholsi were isolated and sequenced, and the corresponding amino acid sequences deduced. The nucleotide sequences of LDH-A of the two populations of G. mirabilis were identical. Five nucleotide differences in the coding region and one amino acid substitution (at position 78) distinguished LDH-As of G. mirabilis and C. nicholsi. The substitution of a glycyl residue (C. nicholsi) for an alanyl residue (G. mirabilis) may account for the difference in thermal stability between these two orthologues. Comparisons of the LDH-A cDNAs of G. mirabilis and G. seta revealed four differences in nucleotide sequence in the coding region, but all nucleotide substitutions were synonymous. The identical deduced primary structures of the two enzymes suggested the possibility of different protein conformational variants ('conformers') in the two species. This hypothesis is supported by electrospray ionization mass spectrometry, which indicates that the masses of the A4-LDH orthologues of the two species are the same within the resolution of the technique. To explore the possibility that the two enzymes were different conformers of the same primary structure, we treated purified G. seta and G. mirabilis A4-LDHs with 3.0 mol l-1 urea or 6 mol l-1 guanidine­HCl and, after removing the denaturant, compared their kinetic properties and heat stabilities. Neither treatment had an effect on the A4-LDH of G. mirabilis, but both converted the Km versus temperature profile of the G. seta enzyme to that of the G. mirabilis A4-LDH. The thermal stability of neither enzyme was affected. We propose, as has been suggested in several previous studies of A4-LDH, that this enzyme can fold into a number of conformers with different stabilities and functional properties. The A4-LDH of G. seta furnishes evidence that such conformers may provide an important mechanism for adaptation of proteins to temperature.