Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus.
Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k(cat) and K(m) values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126.
Suzuki
Toshiharu
T
Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, Japan.
Yasugi
Masako
M
Arisaka
Fumio
F
Oshima
Tairo
T
Yamagishi
Akihiko
A
D050539:3-Isopropylmalate Dehydrogenase; D000220:Adaptation, Biological; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D003080:Cold Temperature; D004789:Enzyme Activation; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase