Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase

Biochemistry
K S Madhusoodanan, R F Colman

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...Continue Reading

References

Nov 15, 1979·Analytical Biochemistry·P A LanzettaO A Candia
Dec 17, 1973·European Journal of Biochemistry·R KobersteinH Sund
Apr 1, 1997·The Journal of Pediatrics·S A WeinzimerP S Thornton

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Citations

Mar 25, 2015·Bioorganic & Medicinal Chemistry Letters·Souta MasudaYasumaru Hatanaka
Dec 17, 2016·Chemical Reviews·György DormánGlenn D Prestwich
Nov 3, 2010·Blood Coagulation & Fibrinolysis : an International Journal in Haemostasis and Thrombosis·Irfan Zia Qureshi, Sobia Kanwal
Sep 27, 2003·The Journal of Biological Chemistry·Tae-Kang KimRoberta F Colman

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