Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase.
Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP. Without activating light, GTPgammaBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgammaBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+-chelate affinity chromatography and reversed-phase HPLC, photolabeled peptides located within or near the phosphate-binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPgammaBP primarily modified a peptide near the middle of the GDH sequence, Asn135-Lys143 and Glu290-Lys295. However, ATPgammaBP modified a single peptide corresponding to the sequence Met411-Arg419 near the C-terminal domain. Using these results and the data from the previously identified base-binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three-dimensional schematic model structure derived from the bacterial enzyme.
Rajagopalan
K
K
Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
Watt
D S
DS
Haley
B E
BE
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D003013:Clostridium / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D010782:Photolysis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017421:Sequence Analysis
D001426:Bacterial Proteins; D001577:Benzophenones; D009711:Nucleotides; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase