Identification of the GTP Binding Site of Human Glutamate Dehydrogenase by Cassette Mutagenesis and Photoaffinity Labeling

Abstract

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 M 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 32 P]GTP was significantly decreased in the presence of 300 M 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 M 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 M, 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. Glutamate dehydrogenase (GDH)1 has been isolated and sequenced from a number of varied sources and has an important role in nitrogen and carbon catabolism. GDH (EC 1.4.1.2-1.4.1.4) catalyzes the reversible deamination of L-glutamate to 2-oxoglutarate using NAD ϩ or NADP ϩ as coenzyme (1). The largest difference between mammalian and bacterial GDH is a long antenna domain formed by the 48-amino acid insertion extending from the top of the NAD domain and lying adjacent to the 3-fold axis of the hexamer (2-5), and there is little identity between the 100 residues in the C terminus (6). In contrast to the extensive allosteric homotrophic and heterotrophic regulation observed in mammalian GDH, bacterial forms of GDH are relatively unregulated. The recent atomic structure of bovine liver GDH suggests that the allosteric regulation and negative cooperativity observed in mammalian GDH may be facilitated by the subunit interactions within the antenna region (4, 5). It was suggested that these allosteric regulations are performed by changing the energy required to open and close the catalytic cleft during enzymatic turnover (4).Mammalian GDH is strictly regulated by several allosteric effectors. GTP inhibits enzyme turnover over a wide range of conditions by increasing the affinity of the enzyme for the product, making product release rate-limiting under all conditions in the presence of GTP (4, 5, 7). In contrast, ADP is a potent activator by decreasing product affinity (8, 9). Binding studies suggest that when NADH is used as a coenzyme, there are two GTP sites per subun...

Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

“…Because we previously identified Lys-450 as a site for GTP binding (26,27), it was not unexpected that the double mutations at 415 and 443 sites within the antenna region of hGDH2 were not influenced to facilitate GTP inhibition. However, as the antenna deletion and a number of the hyperinsulinism-hyper ammonemia mutations have shown (39 -41), the antenna may have a function at least in part to communicate this inhibition.…”

Inhibitory effects of KHG26377 on glutamate dehydrogenase activity in cultured islets

“…GDH activity was measured spectrophotometrically in the direction of reductive amination of 2-oxoglutarate by following the decrease in absorbance at 340 nm (25). All assays were performed in triplicate and initial velocity data were related to a standard assay mixture containing 50 mM triethanolamine, pH 8.0, 100 mM ammonium acetate, 0.1 mM NADH, and 2.6 mM EDTA at 25 o C. One unit of enzyme is defined as the amount required to oxidize 1 μmol of NADH per min at 25 o C. GDH activity was also measured in the direction of oxidative deamination of glutamate (26).…”

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

“…Identification of the nucleotide-binding sites of a variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert into a site upon photoactivation with ultraviolet light. For instance, [ 32 P]2N 3 NAD ϩ was shown to be a valid active site probe for several proteins (23)(24)(25)(26) (27)(28)(29)(30). The ATP-binding site of adenylate kinase and creatine kinase and the protein unique to cerebrospinal fluids of Alzheimer's patients successfully also has been identified using 2N 3 ATP and 8N 3 ATP (31,32).…”