title: Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding.
abstract: Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences. In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively. The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue. In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed. Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Sakowicz
authors.forename: H K
authors.initials: HK
authors.lastname: Kallwass
authors.forename: W
authors.initials: W
authors.lastname: Parris
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: J B
authors.initials: JB
authors.lastname: Jones
authors.forename: M
authors.initials: M
authors.lastname: Gold
mesh_terms: D001483:Base Sequence; D002087:Butyrates / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010667:Phenylpyruvic Acids / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*
chemical_list: D002087:Butyrates; D045782:Hemiterpenes; D007651:Keto Acids; D010667:Phenylpyruvic Acids; D011773:Pyruvates; C015963:2-ketopentanoic acid; D013912:Threonine; C005087:alpha-ketobutyric acid; C001505:alpha-ketoisovalerate; C012375:hydroxypyruvic acid; D007770:L-Lactate Dehydrogenase; C031606:phenylpyruvic acid