Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins.

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degreesC higher melting temperature. Its temperature optimum is 22-25 degreesC higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a "corresponding state" regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

[1]  G Careri,et al.  Statistical time events in enzymes: a physical assessment. , 1975, CRC critical reviews in biochemistry.

[2]  R. Pain,et al.  Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. , 1991, Journal of molecular biology.

[3]  K. Inagaki,et al.  3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization. , 1993, Journal of biochemistry.

[4]  M Vihinen,et al.  Relationship of protein flexibility to thermostability. , 1987, Protein engineering.

[5]  H. Susi.  [22] Infrared spectroscopy—Conformation , 1972 .

[6]  R. Molday,et al.  Substituent effects on amide hydrogen exchange rates in aqueous solution , 1972 .

[7]  A. Hvidt,et al.  Hydrogen exchange in proteins. , 1966, Advances in protein chemistry.

[8]  R. Jaenicke,et al.  Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity. , 1989, European journal of biochemistry.

[9]  A. Hvidt,et al.  Conformational changes in human serum albumin as revealed by hydrogen-deuterium exchange studies. , 1972, The Journal of biological chemistry.

[10]  B. Somogyi,et al.  A double-quenching method for studying protein dynamics: separation of the fluorescence quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors. , 1985, Biochemistry.

[11]  R. Jaenicke,et al.  Proteins under extreme physical conditions , 1990, FEBS letters.

[12]  C. Woodward,et al.  Studies of hydrogen exchange in proteins. 3. The effects of the chymotrypsinogen-alpha chymotrypsin conversion on hydrogen exchange kinetics. , 1970, The Journal of biological chemistry.

[13]  P. Privalov.  Stability of proteins: small globular proteins. , 1979, Advances in protein chemistry.

[14]  T. Tanaka,et al.  High guanine plus cytosine content in the third letter of codons of an extreme thermophile. DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus. , 1984, The Journal of biological chemistry.

[15]  Yawen Bai,et al.  Primary structure effects on peptide group hydrogen exchange , 1993, Biochemistry.

[16]  R. Jaenicke,et al.  Protein stability and molecular adaptation to extreme conditions. , 1991, European journal of biochemistry.

[17]  T. Oshima,et al.  Ligand-induced changes in the conformation of 3-isopropylmalate dehydrogenase from Thermus thermophilus. , 1995, Journal of biochemistry.

[18]  T. Oshima,et al.  Expression, purification, and substrate specificity of isocitrate dehydrogenase from Thermus thermophilus HB8. , 1994, European journal of biochemistry.

[19]  Y. Katsube,et al.  Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. , 1991, Journal of molecular biology.

[20]  S. Lehrer.  Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. , 1971, Biochemistry.

[21]  A. Rosenberg,et al.  Activity and viscosity effects on the structural dynamics of globular proteins in mixed solvent systems , 1989 .

[22]  G. Kohlhaw.  [54] β-Isopropylmalate dehydrogenase from yeast , 1988 .

[23]  R. Jaenicke,et al.  Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. , 1990, Biochemistry.

[24]  S. Parsons,et al.  [108] β-Isopropylmalate dehydrogenase (Salmonella typhimurium) , 1970 .

[25]  N. W. Downer,et al.  Re-examination of rhodopsin structure by hydrogen exchange. , 1982, The Journal of biological chemistry.

[26]  Nobuyuki Fujita,et al.  Systematic sequencing of the Escherichia coli genome: analysis of the 0- 2.4 min region , 1992, Nucleic Acids Res..

[27]  R. Bates,et al.  SECOND DISSOCIATION CONSTANT OF DEUTERIOPHOSPHORIC ACID IN DEUTERIUM OXIDE FROM 5 TO 50. STANDARDIZATION OF A pD SCALE , 1964 .

[28]  M. Eftink,et al.  Exposure of tryptophanyl residues and protein dynamics. , 1977, Biochemistry.

[29]  P. Závodszky,et al.  Hydrogen-exchange study of the conformational stability of human carbonic-anhydrase B and its metallocomplexes. , 1975, European journal of biochemistry.

[30]  K. Miyazaki,et al.  Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8. , 1990, Journal of biochemistry.

[31]  S. Lovett,et al.  Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants. , 1989, Genetics.

[32]  G. Petsko,et al.  Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus. , 1997, Journal of molecular biology.

[33]  A. Szilágyi,et al.  Relationship between thermal stability and 3-D structure in a homology model of 3-isopropylmalate dehydrogenase from Escherichia coli. , 1996, Protein engineering.

[34]  P. Privalov,et al.  Micro- and macro-stabilities of globular proteins , 1979, Nature.