Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling.
It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[gamma-(32)P]azidoguanosine 5'-triphosphate (8-N(3)-GTP). Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-[gamma-(32)P]GTP was significantly decreased in the presence of 300 microm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP. The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.
Lee
E Y
EY
Department of Biochemistry, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Yoon
H Y
HY
Ahn
J Y
JY
Choi
S Y
SY
Cho
S W
SW
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D019920:Photoaffinity Labels; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase