Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 muM 2 -[(4-bromo-2.3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, ph 7.0 and 10 degrees C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (K1=8.5 microM. 5.2 microM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB-[14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5'-AMP and by 0.1 mM 5'-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5'-AMP alone, 5 mM fumarate alone, or 0.1 mM 5'-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB-TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase.

译文

兔肌肉腺苷酸琥珀酸裂解酶与7.5-50 muM 2 -[(4-溴-2.3-二氧丁基) 硫代] 腺苷5 '-单磷酸 (2-BDB-TAMP) 在0.05 M管道缓冲液中孵育,ph 7.0和10摄氏度,给出时间依赖性的双相失活。失活速率表现出对浓度2-BDB-TAMP的非线性依赖性,这可以通过在不可逆反应之前试剂与酶的可逆结合 (K1 = 8.5微米。5.2微米) 来描述,快相和慢相的最大速率常数分别为0.319和0.027 min-1。该酶是四聚体,亚基为50 000 Da。当酶90% 失活时,通过结合蛋白质的磷酸盐分析测得,掺入了0.84摩尔的试剂/摩尔的亚基; 使用2-BDB-[14C]TAMP获得了类似的结果。1毫米5'-AMP和0.1 mM 5 '-AMP + 5毫米富马酸酯 (腺苷酸琥珀酸水解的天然产物) 提供了完全的抗失活和掺入保护,但不是单独使用0.1 mM 5'-AMP,单独使用5毫米富马酸盐,或0.1 mM 5 '-AMP + 5毫米马来酸盐或5毫米琥珀酸盐。这些研究表明2-BDB-TAMP通过底物结合位点的特异性反应使腺苷酸琥珀酸裂解酶失活,亚基之间的负协作性解释了两个失活阶段的出现。用溴化氰裂解2-BDB-TAMP修饰的酶,随后通过反相HPLC分离肽,仅产生一个放射性峰。这种放射性肽用木瓜蛋白酶进一步消化,2-BDB-TAMP反应的靶位点被鉴定为arg112。我们得出结论arg112位于兔肌肉腺苷酸琥珀酸裂解酶的底物结合位点。

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