Collaborative Research: Mechanistic Studies on Fe-type Nitrile Hydration Catalysts

氰化物被广泛用于聚酰胺尼龙等特种化学品的生产。在常温中性水中，腈水合酶(NHASS)催化氰化物水合成酰胺。目前用于氰化物水合成酰胺的工业条件要么使用强酸，要么使用强碱，并且通常与要生产的化合物的敏感结构不相容。因此，氨酶作为制备有机化学中的生物催化剂引起了人们的极大兴趣。尽管NHase酶在工业上具有重要意义，但关于它们如何工作的细节仍然知之甚少。这项研究建议的目的是填补这一知识空白。这一努力的跨学科性质将成为一个平台，以增加妇女和传统上代表性不足的研究生和本科生在生物物理、化学和生物化学领域的科学职业中的参与。作为拟议项目的一部分，两位PI将参与SMART计划，该计划指导密尔沃基地区的公立高中生进行研究项目。通过这一奖项，化学系的生命过程化学计划资助马凯特大学的Richard C.Holz博士和Brian Bennett博士阐明NHase酶的催化机制。将使用一种跨学科的方法，包括动力学、光谱学、生物化学、计算化学以及X射线和中子结晶学。拟议的实验方法旨在解决三个关键问题：i)丁腈水合的过渡态结构是什么？Ii)在整个反应中，具有催化作用的重要物种的质子化状态是什么？以及iii)是什么控制着所谓的真核NHase中的金属分配中心组装？这项研究的成功完成将促进丁腈类手性药物和具有重要工业意义的特种化学品的智能设计和制造，从而极大地造福社会，比目前具有更强的合成灵活性和更低的环境影响。

Nitriles are used extensively in the production of specialty chemicals like the polyamide nylon. Nitrile hydratases (NHases) catalyze the hydration of nitriles to amides at ambient temperatures in neutral water. The currently employed industrial conditions for the hydration of nitriles to amides use either strong acids or bases, and are often incompatible with the sensitive structures of the compounds to be produced. NHases have therefore attracted substantial interest as biocatalysts in preparative organic chemistry. Despite the industrial importance of NHase enzymes, the details of how they work remain poorly understood. The objective of this research proposal is to fill this knowledge gap. The interdisciplinary nature of the effort will serve as a platform to increase the participation of women and classically underrepresented graduate and undergraduate students in scientific careers in biophysics, chemistry, and biochemistry. As part of the proposed project, both PI's will participate in the SMART program that mentors Milwaukee area public high school students in research projects.With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Richard C. Holz and Brian Bennett from Marquette University to elucidate the catalytic mechanism of NHase enzymes. An interdisciplinary approach will be used that will incorporate kinetics, spectroscopy, biochemistry, computational chemistry, and both X-ray and neutron crystallography. The proposed experimental approach is designed to address three key questions: i) What is the transition-state structure of nitrile hydration? ii) What are the protonation states of catalytically important species throughout the reaction? and iii) What controls metallocenter assembly in a purported eukaryotic NHase? The successful completion of this research will greatly benefit society by facilitating the intelligent design and manufacture of nitrile-based chiral pharmaceuticals and industrially important specialty chemicals, with enhanced synthetic flexibility and lower environmental impact, than at present.