A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework.
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
Wilks
H M
HM
Department of Biochemistry, University of Bristol, United Kingdom.
Hart
K W
KW
Feeney
R
R
Dunn
C R
CR
Muirhead
H
H
Chia
W N
WN
Barstow
D A
DA
Atkinson
T
T
Clarke
A R
AR
Holbrook
J J
JJ
D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity
D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase