A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase.
The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.
Department of Chemistry, Syracuse University, New York 13244.
A
A
Banerjee
H R
HR
Levy
G C
GC
Levy
C
C
LiMuti
B M
BM
Goldstein
J E
JE
Bell
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding
D046911:Macromolecular Substances; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase