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首页> 外文期刊>Biochemistry >Implication of His(68) in the substrate site of Bacillus subtilis adenylosuccinate lyase by mutagenesis and affinity labeling with 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5 '-monophosphate
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Implication of His(68) in the substrate site of Bacillus subtilis adenylosuccinate lyase by mutagenesis and affinity labeling with 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5 '-monophosphate

机译：诱变和亲和标记2-[（4-溴-2,3-二氧丁基）硫代]腺苷5'-单磷酸酯酶对枯草芽孢杆菌腺苷琥珀酸裂解酶底物位点His（68）的影响

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Adenylosuccinate lyase of Bacillus subtilis is inactivated by 2-[(4-bromo-2,3-dioxobutyl)thio] adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0. As the reagent concentration is increased, a maximum rate constant is approached, indicative of reversible enzyme-reagent complex formation (K-R = 68 +/- 9 mu M) prior to irreversible modification (k(max) = 0.081 +/- 0.004 min(-1)). Complete inactivation occurs concomitant with about 1 mol of 2-BDB-[C-14]TAMP incorporated/mol of enzyme subunit. Adenylosuccinate, or a combination of AMP and fumarate, decreases the inactivation rate and reduces incorporation of [C-14] reagent, whereas either AMP or fumarate alone is much less effective. These observations suggest that 2-BDB-TAMP attacks the adenylosuccinate binding site. Proteolytic digestion of inactivated enzyme, followed by purification of the digest by HPLC, yields the radioactive peptide Ile(62)-Ala(72), in which Arg(67) and His(68) are the most likely targets. Thus 2-BDB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His(141) attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J. Biol. Chem. 272, 458-465). Site-directed mutagenesis was used to construct mutant enzymes with replacements for both Arg(67) and His(68), and either Arg(67) or His(68). The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay conditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-fold. These results indicate that while Arg(67) and His(68) may both be in the region of the substrate site, only His(68) is important for the catalytic activity of B. subtilis adenylosuccinate lyase. A role is proposed for His(68) as a general acid-base catalyst. [References: 47]
机译：枯草芽孢杆菌的腺苷琥珀酸裂合酶在pH 7.0下被2-[（（4-溴-2,3-二氧代丁基）硫基]腺苷5'-单磷酸酯（2-BDB-TAMP）灭活。随着试剂浓度的增加，将达到最大速率常数，这表明不可逆修饰前的可逆酶-试剂复合物形成（KR = 68 +/- 9μM）（k（max）= 0.081 +/- 0.004 min（ -1））。完全失活伴随着每摩尔酶亚基掺入约1摩尔2-BDB- [C-14] TAMP。腺苷琥珀酸酯或AMP与富马酸酯的组合可降低失活速率并减少[C-14]试剂的掺入，而单独使用AMP或富马酸酯则效果不佳。这些观察结果表明2-BDB-TAMP攻击腺苷琥珀酸酯结合位点。蛋白水解消化灭活的酶，然后通过HPLC纯化消化物，产生放射性肽Ile（62）-Ala（72），其中Arg（67）和His（68）是最可能的靶标。因此，2-BDB-TAMP与腺苷琥珀酸裂合酶在不同于6-BDB-TAMP攻击的His（141）的位点处反应（Lee，Worby，Dixon，and Colman（1997）J. Biol。Chem。272，458-465 ）。定点诱变用于构建突变型酶，取代Arg（67）和His（68）以及Arg（67）或His（68）。在标准测定条件下，R67M突变酶具有与野生型酶几乎相同的比活性，而单个突变H68Q和双重突变R67M-H68Q酶显示的比活性降低了100倍以上。这些结果表明，虽然Arg（67）和His（68）可能都在底物位点区域，但只有His（68）对枯草芽孢杆菌腺苷琥珀酸裂合酶的催化活性很重要。提出了His（68）作为一般酸碱催化剂的作用。 [参考：47]

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