Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.
The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.
Cameron
A D
AD
Department of Chemistry, University of York, Heslington, U.K.
Roper
D I
DI
Moreton
K M
KM
Muirhead
H
H
Holbrook
J J
JJ
Wigley
D B
DB
D000494:Allosteric Regulation; D000596:Amino Acids / Q000502:physiology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005635:Fructosediphosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation
D000596:Amino Acids; D005635:Fructosediphosphates; D009243:NAD; D007770:L-Lactate Dehydrogenase