Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus.
Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.
Fields
Peter A
PA
Biology Department, Franklin and Marshall College, Lancaster, Pennsylvania, USA. peter.fields@fandm.edu
Houseman
Daniel E
DE
D000220:Adaptation, Biological / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000864:Antarctic Regions; D001483:Base Sequence; D003080:Cold Temperature; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D000080864:Lactate Dehydrogenase 5; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010473:Perciformes / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D019289:Pyruvic Acid / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5