title: An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution.
abstract: Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in the absence of Fru(1,6)P(2) under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P(2)-binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P(2) or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P(2) under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P(2)-independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P(2) requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P(2), through a slight decrease and increase of the Fru(1,6)P(2) sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P(2). The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.
authors.affiliation: Department of Applied Biological Science, Science University of Tokyo, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Arai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hishida
authors.forename: Mariko
authors.initials: M
authors.lastname: Ishiyama
authors.forename: Takeo
authors.initials: T
authors.lastname: Kamata
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Uchikoba
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuzawa
authors.forename: Hayao
authors.initials: H
authors.lastname: Taguchi
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008958:Models, Molecular; D011773:Pyruvates / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000235:genetics
chemical_list: D002413:Cations, Divalent; D005635:Fructosediphosphates; D011773:Pyruvates; D011994:Recombinant Proteins; D013912:Threonine; D006639:Histidine; C017092:bromopyruvate; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate