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

Fructose 1,6‐bisphosphate (Fru‐1,6‐P2)‐dependent L‐lactate dehydrogenase (LDH) of Thermus caldophilus GK24 can be converted to a Fru‐1,6‐P2‐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‐P2, 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.