Back to Search
Start Over
Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase.
- Source :
-
Biochimica et biophysica acta [Biochim Biophys Acta] 2001 Feb 09; Vol. 1545 (1-2), pp. 174-83. - Publication Year :
- 2001
-
Abstract
- A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.
- Subjects :
- 3-Isopropylmalate Dehydrogenase
Alcohol Oxidoreductases genetics
Arginine chemistry
Bacillus subtilis enzymology
Bacillus subtilis genetics
Bacterial Proteins genetics
Chemical Phenomena
Chemistry, Physical
Glycine chemistry
Hot Temperature
Leucine chemistry
Models, Molecular
Mutagenesis, Site-Directed
Protein Denaturation
Recombinant Fusion Proteins chemistry
Static Electricity
Thermus thermophilus genetics
Alcohol Oxidoreductases chemistry
Bacterial Proteins chemistry
Thermus thermophilus enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0006-3002
- Volume :
- 1545
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta
- Publication Type :
- Academic Journal
- Accession number :
- 11342043
- Full Text :
- https://doi.org/10.1016/s0167-4838(00)00275-2