The importance of arginine 171 in substrate binding by Bacillus stearothermophilus lactate dehydrogenase

• Published in: Biochemical and biophysical research communications Vol. 146; no. 1; pp. 346 - 353
• Main Authors: Hart, Keith W., Clarke, Anthony R., Wigley, Dale B., Chia, William N., Barstow, David A., Atkinson, Tony, Holbrook, J.John
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 15.07.1987; Elsevier
• Subjects: Amino Acid Sequence; Applied sciences; Arginine; Bacillus stearothermophilus; Binding Sites; Exact sciences and technology; Geobacillus stearothermophilus - enzymology; Hydrogen-Ion Concentration; Kinetics; L-Lactate Dehydrogenase - metabolism; lactate dehydrogenase; Lactates - metabolism; Lactic Acid; Mutation; Other techniques and industries; Pyruvates - metabolism; Pyruvic Acid; Structure-Activity Relationship; Substrate Specificity; LDH; FBP
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(87)90731-5

A variant of lactate dehydrogenase from Bacillus stearothermophilus has been engineered by site-directed mutagenesis in which an active-site arginine residue at position 171 in the protein sequence is replaced by lysine. Replacement of this arginine by lysine has no effect on co-enzyme binding, a relatively small effect on the rate of turnover of the enzyme, but causes a 2000-fold increase in the Michaelis constant for pyruvate, a 6000-fold increase in the dissociation constant for oxamate and results in a Michaelis constant for lactate which is too high to measure. The decrease in binding energy for these carboxylate-containing substrates caused by this mutation is very large, around 5.5 kcal.mol-1 and in part, is explained by the small increase in the distance of a lysine-substrate carboxylate interaction at this site and the absence of the additional hydrogen bond from a two-point arginine-carboxylate interaction. Consistent with this last observation, the ability of this mutant enzyme to stabilize an NAD+-sulphite compound in its active site (an alternative enzyme-substrate complex which does not involve bifurcated bonding to arginine) is only reduced 14-fold.