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: Research Articles; Antarctic; The Antarctic; Antarc*
• Online Access: http://mbe.oxfordjournals.org/cgi/content/short/21/12/2246; 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 <f>(K <rm>m</rm> <rm>PYR</rm>).</f> 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. <f>K <rm>m</rm> <rm>PYR</rm></f> 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°C–20°C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect <f>K <rm>m</rm> <rm>PYR</rm></f> (Gly224Ser, Ala310Pro), and two increased both Ea and <f>K <rm>m</rm> <rm>PYR</rm></f> (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased <f>K <rm>m</rm> <rm>PYR</rm></f> and Ea to levels not significantly different from the A 4 -LDH of a warm temperate fish ( Gillichthys mirabilis , habitat temperature 10°C–35°C). The four single mutants are associated with two α-helices that move during the catalytic cycle; those that affect Ea but not <f>K <rm>m</rm> <rm>PYR</rm></f> 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 ...