Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.
Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.
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混杂酶中的过渡态相互作用：铜绿假单胞菌芳基硫酸酯酶水解硫酸盐和磷酸单酯。
DOI：
10.1021/acs.biochem.8b00996
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Van Loo B
中科院分区：
文献类型：
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作者：
Van Loo B
铜绿假单胞菌芳基硫酸酯酶（PAS）水解硫酸盐和，混杂，磷酸单酯。酶催化的硫酸盐转移在许多生物过程中起着至关重要的作用，但对其催化机理的详细研究还很缺乏。我们提出了线性自由能关系（LFERs）和动力学同位素效应（KIEs）的PAS和活性位点突变体的分析，表明离去基团（LG）稳定的关键作用。在LFER中，PASWT的负布朗斯特系数（β离开基团-Enz = −0.33）比未催化的反应（β离开基团= −1.81）小得多。当阳离子活性位点基团交换为丙氨酸时，这种情况减少。过渡态（TS）过程中的键断裂的程度相当大，证明了一个18 ObridgeKIE为1.0088。几种活性位点突变体的LFER和KIE数据表明，活性位点K375与H211.15N KIE协同作用使离去基团稳定化，并且纯D2 O中硫酸酯酶活性对离去基团能力的敏感性增加（Δβ离去基团H-D = +0.06）表明，随着离去基团能力的降低，从通过刘易斯酸相互作用的电荷补偿到直接质子供给。18 OnonbridgeKIE表明，与未催化的反应相比，PAS催化的硫酸单酯水解的TS具有显著更多的缔合特征，而PAS催化的磷酸单酯水解没有显示出这种变化。酶催化的TS的这种差异似乎是这种混杂水解酶对硫酸盐的特异性优于磷酸酯的主要因素，因为其他特征要么太相似（未催化的TS），要么固有地有利于磷酸盐（电荷）。
Pseudomonas aeruginosaarylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWThas a much less negative Brønsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleaving groupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211.15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S–Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).