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

摘要: By reaction of adenosine 5‘-monothiophosphate with benzophenone-4-maleimide, we synthesized 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 site has not been located in crystal structure hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769−782]. In dark, AMPS-Succ-BP reversibly activates GDH. Irradiation complex at λ >300 nm causes time-dependent, irreversible 2-fold activation enzyme. The kobs for photoactivation shows nonlinear dependence on concentration AMPS-Succ-BP, KR = 4.9 μM kmax 0.076 min-1. photoreaction 20 decreased 10-fold 200 reduced less than NAD, NADH, GTP, or α-ketoglutarate. Modified no longer still inhibited GTP hi...