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首页> 外文期刊>Biochemistry >Adenosine 5'-O-[S-(4-Succinimidyl-benzophenone)thiophosphate]:A New Photoaffinity Label of the Allosteric ADP Site of Bovine Liver Glutamate Dehydrogenase
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Adenosine 5'-O-[S-(4-Succinimidyl-benzophenone)thiophosphate]:A New Photoaffinity Label of the Allosteric ADP Site of Bovine Liver Glutamate Dehydrogenase

机译：腺苷5'-O- [S-（4-琥珀酰亚胺基-二苯甲酮）硫代磷酸酯]：牛肝谷氨酸脱氢酶变构ADP位点的新光亲和标记

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摘要

By reaction of adenosine 5’—monothiophosphate with benzophenone-4-maleimide, we synthe-sized adenosine S’-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 2 >300 nm causes a time-dependent, ilTeversible 2-fold activation of the enzyme. The k0b~ for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with KR = 4.9 ~M and kmax = 0.076 min’. The k0b~ for photoreaction by 20 fIM AMPS-Succ-BP is decreased 10-fold by 200 ~iM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or u-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 [3H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys488—Glu495 has been identified as the only reaction target, and the data suggest that Arg49’ is the modified amino acid. Arg49’ (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.
机译：通过使腺苷5'-单硫代磷酸酯与二苯甲酮-4-马来酰亚胺反应，我们合成了大小适中的腺苷S'-O- [S-（4-琥珀酰亚胺基-二苯甲酮）硫代磷酸酯]（AMPS-Succ-BP）作为光反应性ADP类似物。已知牛肝谷氨酸脱氢酶被ADP变构激活，但ADP位点尚未位于六聚酶的晶体结构中[Peterson，P. E.和Smith，T. J.（1999）Structure 7，769-782]。在黑暗中，AMPS-Succ-BP可逆地激活GDH。谷氨酸脱氢酶和AMPS-Succ-BP的复合物在2> 300 nm处辐照会引起该酶的时间依赖性不可逆2倍活化。用于光活化的k0b〜显示出对AMPS-Succ-BP浓度的非线性依赖性，KR = 4.9〜M，kmax = 0.076 min'。通过20 fIM AMPS-Succ-BP进行光反应的k0b〜被200μMADP降低了10倍，但被NAD，NADH，GTP或u-酮戊二酸降低了不到2倍。修饰的酶不再被ADP激活，但仍被GTP和高浓度的NADH抑制。这些结果表明AMPS-Succ-BP的反应发生在ADP位点内。该酶包含最多0.5 mol [3H] AMPS-Succ-BP / mol酶亚基或3 mol试剂/ mol六聚体。 Lys488-Glu495肽已被鉴定为唯一的反应靶标，数据表明Arg49'是修饰的氨基酸。因此，认为Arg49'（在靠近GDH的GTP＃2结合结构域的C末端螺旋中）位于酶的变构ADP位点或附近。根据这些结果，将AMPS-Succ-BP置于谷氨酸脱氢酶的晶体结构中，并在其中标记该酶的ADP结合位点。

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