A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: comparison with a structured monomeric intermediate obtained during refolding.
The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site-directed mutagenesis of C. symbiosum gdh gene was performed to replace the residues Arg-61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over-expression in Escherichia coli of the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross-reacts with an anti-wild-type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.
Millevoi
S
S
Dipartimento di Scienze Biochimiche, A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
Pasquo
A
A
Chiaraluce
R
R
Consalvi
V
V
Giangiacomo
L
L
Britton
K L
KL
Stillman
T J
TJ
Rice
D W
DW
Engel
P C
PC
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D005969:Glutamate Dehydrogenase; D019791:Guanidine