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Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3‐isopropylmalate dehydrogenase
Author(s) -
Németh Attila,
Svingor Ádám,
Pócsik Márta,
Dobó József,
Magyar Csaba,
Szilágyi András,
Gál Péter,
Závodszky Péter
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01190-x
Subject(s) - thermostability , thermus thermophilus , thermophile , glutamate dehydrogenase , enzyme , thermus , biochemistry , chemistry , mesophile , escherichia coli , mutant , glutamine , amino acid , stereochemistry , biology , crystallography , bacteria , glutamate receptor , genetics , receptor , gene
The comparison of the three‐dimensional structures of thermophilic ( Thermus thermophilus ) and mesophilic ( Escherichia coli ) 3‐isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.