Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus.
A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability.
Yasugi
M
M
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan.
Suzuki
T
T
Yamagishi
A
A
Oshima
T
T
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D004355:Drug Stability; D015723:Gene Library; D007700:Kinetics; D009154:Mutation; D013696:Temperature; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase