AMINO-ACID-SEQUENCE DIFFERENCES CANNOT FULLY EXPLAIN INTERSPECIFIC VARIATION IN THERMAL SENSITIVITIES OF GOBIID FISH A(4)-LACTATE DEHYDROGENASES (A(4)-LDHS)

We compared the deduced amino acid sequences, heat stabilities and the rmal sensitivities of a kinetic property, the apparent Michaelis-Mente n constant (K-m) of pyruvate, of A(4)-lactate dehydrogenase (A(4)-LDH) in four species of goby fishes (Family Gobiidae), adapted to differen t temperatures, to examine how changes in primary structure influence the adaptation of enzymes. The effect of temperature on K-m of pyruvat e reflected each species' environmental temperature. For the most eury thermal species, Gillichthys seta, which is endemic to shallow interti dal regions of the upper Gulf of California and encounters temperature s between approximately 9 and 40 degrees C, K-m of pyruvate was minima lly affected by temperature, compared with the A(4)-LDH orthologues fr om a less eurythermal congener, G. mirabilis (9-30 degrees C), a cold temperate goby, Coryphopterus nicholsi (10-18 degrees C) and a tropica l species, C. personatus (25-32 degrees C). Heat denaturation profiles failed to correlate with habitat temperature; G. mirabilis A(4)-LDH w as most thermally stable, followed by the orthologues of C. nicholsi a nd G. seta. Complementary DNAs (cDNAs) encoding LDH-As of G. seta, Gul f of California and Pacific coast populations of G. mirabilis and C. n icholsi mere isolated and sequenced, and the corresponding amino acid sequences deduced. The nucleotide sequences of LDH-A of the two popula tions of G. mirabilis were identical. Five nucleotide differences in t he coding region and one amino acid substitution (at position 78) dist inguished LDH-As of G. mirabilis and C. nicholsi. The substitution of a glycyl residue (C. nicholsi) for an alanyl residue (G. mirabilis) ma y account for the difference in thermal stability between these two or thologues. 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 deduc ed primary structures of the two enzymes suggested the possibility of different protein conformational variants ('conformers') in the two sp ecies. This hypothesis is supported by electrospray ionization mass sp ectrometry, which indicates that the masses of the A(4)-LDH orthologue s of the two species are the same within the resolution of the techniq ue. To explore the possibility that the two enzymes were different con formers of the same primary structure, we treated purified G. seta and G. mirabilis A(4)-LDHs with 3.0 mol l(-1) urea or 6 mol l(-1) guanidi ne-HCl and, after removing the denaturant, compared their kinetic prop erties and heat stabilities. Neither treatment had an effect on the A( 4)-LDH of G. mirabilis, but both converted the Km versus temperature p rofile of the G. seta enzyme to that of the G. mirabilis A(4)-LDH. The thermal stability of neither enzyme was affected. We propose, as has been suggested in several previous studies of A(4)-LDH, that this enzy me can fold into a number of conformers with different stabilities and functional properties. The A(4)-LDH of G. seta furnishes evidence tha t such conformers may provide an important mechanism for adaptation of proteins to temperature.