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Identification of an allosteric site residue of a fructose 1,6‐bisphosphate‐dependent L‐lactate dehydrogenase of Thermus caldophilus GK24: production of a non‐allosteric form by protein engineering
Author(s) -
Matsuzawa Hiroshi,
Machida Masayuki,
Kunai Kenji,
Ito Yoshiyuki,
Ohta Takahisa
Publication year - 1988
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 - 10.1016/0014-5793(88)80464-2
Subject(s) - allosteric regulation , biochemistry , chemistry , residue (chemistry) , trypsin , lactate dehydrogenase , dehydrogenase , enzyme
Fructose 1,6‐bisphosphate (Fru‐1,6‐P 2 )‐dependent L‐lactate dehydrogenase (LDH) of Thermus caldophilus GK24 can be converted to a Fru‐1,6‐P 2 ‐independent form on modification with Arg‐specific reagents. After trypsin digestion of the modified LDH, a peptide containing a modified Arg residue was purified and sequenced, and the modified Arg residue was identified as Arg‐173. Subsequently, Arg‐173 was replaced with Gln to remove the positive charge by site‐directed mutagenesis. The mutant LDH was independent of Fru‐1,6‐P 2 , like non‐allosteric vertebrate LDHs. Thus, Arg‐173 was concluded to be located in the allosteric site and to be responsible for allosteric regulation of the LDH.