Interactions of macromolecular crowding agents and cosolutes with small-molecule substrates: effect on horseradish peroxidase activity with two different substrates.
Interactions of macromolecular crowding agents and cosolutes with small-molecule substrates: effect on horseradish peroxidase activity with two different substrates.
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DOI：
10.1021/jp506594f
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发表时间：
2014-09-11
期刊：
影响因子：
--
通讯作者：
Keating CD
中科院分区：
文献类型：
--
作者：
Aumiller WM Jr;Davis BW;Hatzakis E;Keating CD
溶液组成对酶反应的重要性日益受到重视，特别是对于大分子共溶体。大分子拥挤及其对酶反应的影响已经被研究过几种酶，通常被理解为酶构象的变化。在拥挤溶液中，酶反应的小分子底物的化学性质相对较少受到关注。本文研究了辣根过氧化物酶(HRP)与两种疏水性不同的小分子底物的反应。当底物为疏水性的3，3‘，5，5’-四甲基联苯胺(TMB)和亲水性更强的邻苯二胺(OPD)时，挤压剂和共溶体对HRP活性的影响有很大不同。与TMB的反应速率对排泄剂和共溶物的存在比OPD更敏感，这表明小分子底物本身可能与排泄物和共溶物相互作用。在较高的聚乙二醇组分(25-30wt/wt%)下，TMB未发生反应。即使在较低的浓度下，疏水性较强的底物对HRP的米氏常数(Km)在拥挤剂和共溶质的存在下也会增加，尤其是在聚乙二醇的存在下。用脉冲梯度核磁共振技术研究了TMB和OPD在聚乙二醇和葡聚糖反应介质中的扩散行为。在10%PEG8K溶液中，TMB的扩散系数比在缓冲液中降低了3.9倍，而在OPD中仅降低了1.7倍。综上所述，这些数据表明，小分子底物与聚集物或共溶质之间的弱吸引力相互作用会降低底物的化学活性，从而降低酶的活性，并且这些影响因所涉及的分子的特性而异。由于许多酶可以作用于多种底物，因此在理解复杂介质(如生物液)中的酶反应时，考虑底物化学是很重要的。
The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25–30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.
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