Adenosine 5‘-O-[S-(4-Succinimidyl-benzophenone)thiophosphate]:  A New Photoaffinity Label of the Allosteric ADP Site of Bovine Liver Glutamate Dehydrogenase

Abstract

By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.

Critical Role of Lys212 and Tyr140 in Porcine NADP-dependent Isocitrate Dehydrogenase

“…␣-Ketoglutarate, sodium salt (0.186 mg, 1 mmol), was dissolved in 0.6 ml of water; the pH of the solution was adjusted to around 8.0, and the volume of the solution was brought up to 1.0 ml with distilled water to yield a 1 M solution. An aliquot (100 l) of the 1 M ␣-ketoglutarate solution was added to 1.0 ml of The concentration of [ 3 H]␣-ketoglutarate was determined by using the compound as a substrate for glutamate dehydrogenase, as described previously (17). Briefly, bovine liver glutamate dehydrogenase was diluted with 0.1 M potassium phosphate buffer, pH 7.1.…”

“…Briefly, bovine liver glutamate dehydrogenase was diluted with 0.1 M potassium phosphate buffer, pH 7.1. Various concentrations of ␣-ketoglutarate (from 10 M to 3 mM) were added to an assay solution that contained 10 mM Tris acetate buffer, pH 8, with 10 M EDTA, 100 M NADH, and 50 mM ammonium chloride (17). The glutamate dehydrogenase activity was measured by the rate of oxidation of NADH to NAD at 340 nm and 25°C.…”

Insights from Affinity Labeling into Nucleotide- and Coenzyme-Dependent Enzyme Catalysis and Regulation

“…The benzophenone's carbonyl group, which is the locus of the photoreaction, will likely be positioned to react with an amino acid outside the normal ADP binding site. However, identification of the modified amino acid residue may allow one to deduce the location of the actual ADP binding site [38]. Given the size of AMPS-Succ-BP, it may be more suitable for affinity labeling of NAD sites in enzymes.…”

“…Rather, the product of covalent reaction between the enzyme and periodate-oxidized nucleotide is more likely to be a dihydroxymorpholino derivative, which is unstable under conditions of proteolytic digestion [6]. Very few labeled peptides have been isolated from enzymes modified by , which features an adenosine 5'-monothiophosphate group linked to the photoreactive benzophenone through the succinimidyl ring [38]. AMPS-Succ-BP has key structural elements of ADP (the adenine, ribose and one phosphate, with a negative charge at neutral pH) so that it can be recognized by an ADP binding site.…”

Structure-assisted ligand-binding analysis using fluorogenic photoaffinity labeling

“…These results are consistent with previous results of PAL with AMPS-Succ-BP. 22 Alternatively, these inhibition effects could be studied using SDS-PAGE by monitoring the fluorescence due to the presence of coumarin (Fig. S6).…”