Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.
The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.
Kochhar
S
S
Nestlé Research Centre, Lausanne, Switzerland; European Molecular Biology Laboratory, Hamburg, Germany. sunil.kochhar@rdls.nestle.com
Lamzin
V S
VS
Razeto
A
A
Delley
M
M
Hottinger
H
H
Germond
J E
JE
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D006639:Histidine / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D045022:Lactate Dehydrogenases; D007778:Lactobacillus / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000502:physiology*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D001224:Aspartic Acid; D006639:Histidine; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase