Enzyme cooperativity, biocatalysis and bioconjugation

酶不仅是令人着迷的催化剂，也是大多数药物的靶标。由于其复杂的三维结构，酶显示出许多化学催化剂中罕见的有趣特性。细胞色素P450酶(Cyps或P450)是在所有王国中普遍存在的酶，涉及到药物代谢、天然产物的生物合成、类固醇、脂质等过程。已知一些P450(例如，CYP3A4)表现出变构效应和/或使用一种独特的协同作用机制，涉及同时与酶结合的相同或不同配体的多个拷贝之间的直接相互作用。使用现有的方法来描述这样的行为是相当具有挑战性的。为了研究CYP3A4的变构和协同作用，我们开发了一种新的生物偶联方法和一些即将应用的生物物理方法。我们目前还在开发一种使用等温量热法的动力学方法。*在合成化学中使用酶是有利的，不仅因为它与环境友好的条件兼容，还因为许多酶催化化学催化剂无法实现的反应。例如，我们对P450感兴趣，因为它们能够在未活化的C-H键上催化选择性羟基化。几丁质酶和木质素分解过氧化物酶也是我们感兴趣的，因为它们可以分别降解一些最坚韧和最丰富的底物，几丁质和木质素。我们建议开发新的方法来促进生物催化的应用。例如，我们率先使用化学助剂来控制和预测P450催化反应的区域和立体选择性。使用一种带有大的杂乱底物结合口袋的酶，我们最近证明了通过合理的突变来限制活性部位可以显著改变酶的选择性。最后，我们收集了初步的数据，证明了机械力化学可以积极地影响酶过程，甚至超过当前的化学工业过程。我们建议在感兴趣的特定过程中应用和优化这些方法。*该研究计划的总体目标是开发新的工具来研究酶并将其应用于酶学，并促进生物催化。具有代表性的具体目标包括：*-应用我们的生物物理方法研究氨基糖苷3‘-磷酸转移酶*-在动力学模式下使用等温量热法研究P450*-使用我们的新的CyP3A4生物偶联物并生成新的生物偶联物来研究CYP3A4的变构和协同作用*-寻找新的化学辅助剂来控制P450的区域和立体选择性*-应用、优化和研究我们的机械化学方法与新的酶反应

Enzymes are not only fascinating catalysts, but they are also the target of most drugs. Thanks to their complex 3D structure, enzymes show many interesting characteristics rarely found in chemical catalysts.******Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous enzymes found across all kingdoms and involved in processes as varied as drug metabolism, biosynthesis of natural products, steroids, lipids, and more. Some P450s (e.g. CYP3A4) are known to show allosteric effects and/or use a unique cooperativity mechanism involving direct interactions between multiple copies of the same or different ligands bound to the enzyme at the same time. Such a behavior is quite challenging to delineate using existing methods. In order to study the allostery and cooperativity of CYP3A4, we have developed a new bioconjugation approach and a number of biophysical methods which will be applied. We are also currently developing a kinetic method using isothermal calorimetry.******The use of enzymes in synthetic chemistry is advantageous not only because it is compatible with environmentally benign conditions but also because many enzymes catalyze reactions that are inaccessible to chemical catalysts. For example, P450s are of interest to us because of their ability to catalyze selective hydroxylations at unactivated C-H bonds. Chitinases and lignolytic peroxidases are also of interest to us because they can degrade some of the toughest and most abundant substrates, chitin and lignin respectively. We propose to develop new methods to facilitate biocatalytic applications. For example, we have pioneered the use of chemical auxiliaries to control and predict the regio- and stereo-selectivity of P450-catalyzed reactions. Using an enzyme with a large promiscuous substrate binding pocket, we have recently demonstrated that constraining the active site via rational mutagenesis can dramatically shift the selectivity of the enzyme. Finally, we have gathered preliminary data demonstrating that mechanochemistry can positively affect enzymatic processes and even surpass current chemical industrial processes. We propose to apply and optimize these methods for specific processes of interest.******The overall goals of this research program are to develop new tools to study enzymes and apply these in enzymology and to facilitate biocatalysis. Representative specific aims include:******- Apply our biophysical methods to study aminoglycoside 3'-phosphoryltransferase***- Study P450 using isothemal calorimetry in kinetics mode***- Use our new CYP3A4 bioconjugates and generate new ones to study CYP3A4 allostery and cooperativity***- Identify new chemical auxiliaries to control the regio- and stereo-selectivities of P450s***- Apply, optimize and investigate our mechanochemical method with new enzymatic reactions