Decreases in Activation Energy and Substrate Affinity in Cold Adapted A4-Lactate Dehydrogenase: Evidence from the Antarctic Notothenioid Fish Chaenocephalus Aceratus

• Published in: Molecular Biology and Evolution
• Main Authors: Fields, Peter A., Houseman, Daniel E.
• Format: Text
• Language: English
• Published: Oxford University Press 2004
• Subjects: Original Article; Antarctic; The Antarctic; Antarc*
• Online Access: http://mbe.oxfordjournals.org/cgi/content/short/msh237v1; https://doi.org/10.1093/molbev/msh237

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°C), and orthologs of the enzyme A 4 -lactate dehydrogenase (A 4 -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 ( K m PYR). Here, site directed mutagenesis was used to determine which amino acid substitutions found in A 4 -LDH of the notothenioid Chaenocephalus aceratus , with respect to orthologs from warm adapted teleosts, are responsible for these adaptive changes in enzyme function. K m 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 - 20°C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect K m PYR (Gly224Ser, Ala310Pro), and two increased both Ea and K m PYR (Glu233Met, Gln317Val). The double mutants Glu233Met / Ala310Pro and Glu233Met / Gln317Val increased K m PYR and Ea to levels not significantly different from the A 4 -LDH of a warm temperate fish ( Gillichthys mirabilis , habitat temperature 10 - 35°C). The four single mutants are associated with two α- helices that move during the catalytic cycle; those that affect Ea but not K m PYR are further from the active site than those that affect both parameters. These results provide evidence that: 1) cold adaptation in A 4 -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 α helices, perhaps due to differences in proximity to the active site.