Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces.
X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.
Dean
J L
JL
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Cölfen
H
H
Harding
S E
SE
Rice
D W
DW
Engel
P C
PC
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; D005969:Glutamate Dehydrogenase