Hye-Young Yoon

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 μmfor 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 μmGTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[γ-32P]azidoguanosine 5′-triphosphate (8-N3-GTP). Saturation of photoinsertion with 8-N3-GTP occurred apparent K d values near 20 μm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N3-[γ-32P]GTP was significantly decreased in the presence of 300 μm 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 μm 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 μm, 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.

TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells.

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.

A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P3 binding. Here, we tested the hypothesis that PtdIns(3,4,5)P3-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P3, but with relatively low affinity (≈1.6 μm), and the PH domains did not mediate PtdIns(3,4,5)P3-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P3 binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P3, the subsequent production of which triggers enzymatic activity.

Identification of an NAD+ Binding Site of Brain Glutamate Dehydrogenase Isoproteins by Photoaffinity Labeling

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The Xindicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.

Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase.

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD+ binding site within human GDH encoded by the synthetic human GDH gene and expressed inEscherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+ binding site. Amino acid sequencing and compositional analysis identified Glu279 as the site of photoinsertion into human GDH, suggesting that Glu279 is located at or near the NAD+ binding site. The importance of the Glu279 residue in the binding of NAD+ was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu279 has no effects on the expression or stability of the different mutants. The K m values for NAD+ were 10–14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V maxvalues were similar for wild-type and mutant GDHs. The efficiency (k cat/K m ) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K m values for NAD+. In contrast to the K m values for NAD+, wild-type and mutant GDHs show similarK m values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu279 is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu279 plays an important role for efficient binding of NAD+ to human GDH.

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking.

Background information. ARAP1 is an Arf (ADP‐ribosylation factor)‐directed GAP (GTPase‐activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1.

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

UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD+-binding site within human UGDH by photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3 NAD+), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+-binding site. Amino acid sequencing and compositional analysis identified the NAD+-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD+ was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [32P]2N3NAD+ was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD+ to human UGDH.

Photoaffinity Labeling of Brain Glutamate Dehydrogenase Isoproteins with an Azido-ADP

The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [α-32P]8-azidoadenosine 5′-diphosphate (8N3ADP). 8N3ADP, without photolysis, mimicked the activatory properties of ADP on GDH I and GDH II activities, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [α-32P]8N3ADP occurred at around 40∼50 μm photoprobe with apparent K d values near 25 and 40 μm for GDH I and GDH II, respectively. Photoinsertion of [α-32P]8N3ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affinity chromatography and reversed-phase high performance liquid chromatography, photolabel-containing peptides generated by tryptic digestion were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as in the region containing the sequence, EMSWIADTYASTIGHYDIN. Photolabeling of the peptide was prevented over 90% by the presence of 1 mm ADP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as ADP. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide with the residues Glu179–Asn197 is within the ADP binding domain of the brain GDH isoproteins.

Effects of ADP on different inhibitory properties of brain glutamate dehydrogenase isoproteins by perphenazine.

Incubation of glutamate dehydrogenase isoproteins (GDH I and GDH II) from bovine brains with perphenazine resulted in a time-dependent loss of enzyme activity. 2-Oxoglutarate and NADH, separately or together, gave partial but not complete protection against the inhibition. Although there were no detectable differences between GDH I and GDH II in inhibition by perphenazine in the absence of ADP, the sensitivities to the inhibition by the drug were significantly distinct for the two isoproteins in the presence of ADP. Low concentrations of ADP (0.05-0.20 mM) did not interfere with the inhibition of GDH I and GDH II by perphenazine. However, in the presence of high concentrations of ADP (0.5-1.0 mM), inhibitory effects of perphenazine on GDH isoproteins were significantly diminished as determined by enzyme kinetics and quantitative affinity chromatography on perphenazine-Sepharose. GDH I was more sensitively reacted with ADP than GDH II on the inhibition by perphenazine. Since physiological ADP levels can vary from 0.05 to > 1.0 mM depending on the rate of oxidative phosphorylation, our results suggest a possibility that two types of GDHs are differently regulated by the antipsychotic actions of perphenazine depending on the physiological concentrations of ADP. GTP and L-leucine, other well-known allosteric regulators, did not affect the inhibitory actions of perphenazine on bovine brain GDH isoproteins.