The A245K Mutation Exposes Another Stage of the Bacterial l-Lactate Dehydrogenase Reaction Mechanism

• Published in: Biochemistry (Easton) 2001-06, Vol.40 (24), p.7247-7252
• Main Authors: Kȩdzierski, Paweł, Moreton, Kathleen, Clarke, Antony R, Holbrook, J. John
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Bacillus stearothermophilus; Binding Sites - genetics; Catalysis; Computer Simulation; drug design; Electron Transport; Escherichia coli; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Kinetics; L-Lactate Dehydrogenase - chemistry; L-Lactate Dehydrogenase - genetics; L-Lactate Dehydrogenase - metabolism; Lysine - genetics; Models, Chemical; Mutagenesis, Site-Directed; NAD - metabolism; Protein Conformation; Solvents; Substrate Specificity - genetics; Viscosity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0026775

The A245K mutant of Bacillus stearothermophilus l-lactate dehydrogenase has been expressed in Escherichia coli and purified. A qualitative change in the reaction mechanism prior to the hydride transfer step in the reverse direction in the mutant is revealed. Both transient and steady state characteristics of the mutant are presented and show in contrast to the wild-type enzyme where a rearrangement of an enzyme−NADH−pyruvate complex is rate-limiting that in the mutant the rearrangement is much faster and hydride transfer is the first slow step. The steady state is limited by a new second slower conformation change involving an NAD+ complex. The mutation may provide a valuable framework for inhibitor and drug design research.