Adenosine 5'-O-[S-(4-Succinimidyl-benzophenone)thiophosphate]: A New Photoaffinity Label of the Allosteric ADP Site of Bovine Liver Glutamate Dehydrogenase
详细信息 查看全文
- 作者：K. S. Madhusoodanan and Roberta F. Colman
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：February 13, 2001
- 年：2001
- 卷：40
- 期：6
- 页码：1577 - 1586
- 全文大小：339K
- 年卷期：v.40,no.6(February 13, 2001)
- ISSN：1520-4995
文摘
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 photoreactiveADP 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 
>300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The kobs for photoactivation shows nonlineardependence on the concentration of AMPS-Succ-BP, with KR = 4.9 
M and kmax = 0.076 min-1. The kobsfor photoreaction by 20 M AMPS-Succ-BP is decreased 10-fold by 200 M ADP, but is reduced lessthan 2-fold by NAD, NADH, GTP, or 
-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 ofAMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [3H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys488-Glu495 has beenidentified as the only reaction target, and the data suggest that Arg491 is the modified amino acid. Arg491(in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or nearthe enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned withinthe crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of theenzyme.
>300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The kobs for photoactivation shows nonlineardependence on the concentration of AMPS-Succ-BP, with KR = 4.9 M and kmax = 0.076 min-1. The kobsfor photoreaction by 20 M AMPS-Succ-BP is decreased 10-fold by 200 M ADP, but is reduced lessthan 2-fold by NAD, NADH, GTP, or -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 ofAMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [3H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys488-Glu495 has beenidentified as the only reaction target, and the data suggest that Arg491 is the modified amino acid. Arg491(in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or nearthe enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned withinthe crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of theenzyme.