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°C), and orthologs of the enzyme A...
| Published in: | Molecular Biology and Evolution |
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| Main Authors: | , |
| Format: | Text |
| Language: | English |
| Published: |
Oxford University Press
2004
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| Subjects: | |
| Online Access: | http://mbe.oxfordjournals.org/cgi/content/short/21/12/2246 https://doi.org/10.1093/molbev/msh237 |
| Summary: | 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 ... |
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