Dynamics of a mobile loop at the active site of Escherichia coli asparaginase
Dynamics of a mobile loop at the active site of Escherichia coli asparaginase
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DOI：
10.1016/s0167-4838(00)00179-5
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发表时间：
2000-09-29
期刊：
影响因子：
--
通讯作者：
Röhm, KH
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文献类型：
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作者：
Aung, HP;Bocola, M;Röhm, KH
来自大肠杆菌的天冬酰胺酶II是众所周知的细菌II类酰胺水解酶的成员。该家族的酶利用一种特殊的催化机制，其中一对苏氨酸残基起关键作用。另一个共同的特征是当底物结合时在活性位点上闭合的移动的表面环。我们用色氨酸残基的荧光作为光谱探针，通过停流实验研究了环的运动。使用野生型酶，监测唯一色氨酸W 66的荧光。天冬酰胺在这里诱导环的快速闭合。该过程的速率常数（100-150 s（-1），在4 ℃）显著高于限速催化步骤的速率常数。通过用酪氨酸取代W 66和用色氨酸取代Y25（环的一个组分）来产生更具选择性的光谱探针。在所得酶变体中，k（cat）和环运动速率分别降低10（2）和> 10（3）倍，而底物结合不受影响。这表明25位酪氨酸的存在对于闭环和催化都是必不可少的。所观察到的瞬变的数值模拟是一致的，与一个模型，环路闭合是一个绝对的先决条件，基板营业额。(C)2000 Elsevier Science B. V.保留所有权利。
Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wildtype enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and > 10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover. (C) 2000 Elsevier Science B.V. All rights reserved.