The importance of arginine 171 in substrate binding by Bacillus stearothermophilus lactate dehydrogenase.
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.
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D009154:Mutation; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007773:Lactates; D011773:Pyruvates; D019344:Lactic Acid; D019289:Pyruvic Acid; D001120:Arginine; D007770:L-Lactate Dehydrogenase