An investigation of the contribution made by the carboxylate group of an active site histidine-aspartate couple to binding and catalysis in lactate dehydrogenase.
The influence of aspartate-168 on the proton-donating and -accepting properties of histidine-195 (the active site acid/base catalyst in lactate dehydrogenase) was evaluated by use of site-directed mutagenesis to change the residue to asparagine and to alanine. Despite the fact that asparagine could form a hydrogen bond to histidine while alanine could not, the two mutant enzymes have closely similar catalytic and ligand-binding properties. Both bind pyruvate and its analogue (oxamate) 200 times more weakly than the wild-type enzyme but show little disruption in their binding of lactate and its unreactive analogue, trifluorolactate. Neither mutation alters the binding of coenzymes (NADH and NAD+) or the pK of the histidine-195 residue in the enzyme-coenzyme complex. We conclude that a strong histidine-aspartate interaction is only formed when both coenzyme and substrate are bound. Deletion of the negative charge of aspartate shifts the equilibrium between enzyme-NADH-pyruvate (protonated histidine) and enzyme-NAD+-lactate (unprotonated histidine) toward the latter. In contrast to the wild-type enzyme, the rate of catalysis in both directions in the mutants is limited by a slow hydride ion transfer step.
Clarke
A R
AR
Department of Biochemistry, University of Bristol Medical School, U.K.
Wilks
H M
HM
Barstow
D A
DA
Atkinson
T
T
Chia
W N
WN
Holbrook
J J
JJ
D001224:Aspartic Acid; D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006639:Histidine; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation
D001224:Aspartic Acid; D006639:Histidine; D007770:L-Lactate Dehydrogenase