Metal requirement and mechanism of anthrax lethal factor and other zinc proteases

锌是一种必需元素，也是参与许多关键生物过程的300多种酶的不可或缺的组成部分。我的研究计划试图在分子水平上了解这些酶是如何工作的。我们的主要研究重点是炭疽致死因子(LF)，这是一种依赖于锌的金属酶，它构成了炭疽杆菌分泌的炭疽毒素的三种成分之一。尽管人们普遍认为LF是一种锌酶(即它需要锌来发挥作用)，但令人惊讶的是，它对钴、镍或铜的活性更高。我们拟议的工作将利用这一特征，通过对金属取代的酶形式进行详细的光谱研究，在分子水平上了解LF是如何工作的。铜形式的LF将特别重要，不仅因为它是所有金属取代的LF变体中最活跃的，还因为它的高活性是如此不寻常(大多数锌酶的活性非常低)。此外，在适当的条件下，铜能否激活其他锌酶，如热溶酶(一种大规模用于生产甜味剂阿斯巴甜的酶)或羧肽酶A(一种重要的消化酶)，将进行调查。了解一种金属为何(以及在何种条件下)比另一种金属表现更好，对于改进现有催化剂和设计新型催化剂至关重要。此外，酪氨酸残基在LF(Tyr728)中的作用将被研究，这对LF的功能非常重要，以提供更好的了解该酶的机制。拟议工作的第二个方面在于表征LF中的第二个锌结合位点。锌(或其他金属)占据这个位置会抑制该酶，但最近也被证明加速了一个金属离子与另一个金属离子交换的分子过程(例如，用锌取代铜)。拟议的研究将阐明第二种金属是如何与Lf结合的，以及在其他锌酶中发现的类似金属结合部位是否也可以促进金属交换反应。因此，我们的工作将有助于更好地了解金属交换反应的机制，这些反应在许多关键的生物过程中都是重要的。最后，我们建议探索将锌结合时变色的化合物作为锌可及性探针(ZAP)用于LF和其他锌酶的结构研究的可能性。从这些研究中获得的见解可能会导致开发新的工具来探索金属蛋白的结构方面。综上所述，拟议的研究将进一步加深我们对金属在LF和其他锌酶机制中的作用的理解。这些见解可能被用来开发针对LF和其他结构相关的锌酶的新疗法，如肉毒杆菌和破伤风神经毒素。

Zinc is an essential element, and an indispensable constituent of more than 300 enzymes involved in many critical biological processes. My research program seeks to understand how these enzymes work on the molecular level. Our primary focus of study is the anthrax lethal factor (LF), a zinc-dependent metalloenzyme, which constitutes one of the three components of the anthrax toxin secreted by Bacillus anthracis.Although it is generally accepted that LF is a zinc enzyme (i.e., it requires zinc for function), it is surprisingly more active with cobalt, nickel or copper. Our proposed work will exploit this feature to gain an understanding of how LF works at the molecular level by performing detailed spectroscopic studies on the metal-replaced forms of the enzyme. The copper form of LF will be of particular importance, not only because it is the most active of all metal-substituted LF variants but also because its high activity is so unusual (the majority of zinc enzymes have very low activity). In addition, whether other zinc enzymes such as thermolysin (an enzyme used on a large scale in the production of the sweetener aspartame) or carboxypeptidase A (an important digestive enzyme) can be activated by copper given the proper conditions will be investigated. Knowledge on why (and under which conditions) one metal can perform better than another one is critical to improving existing and designing novel catalysts. Furthermore, the role of a tyrosine residue in LF (Tyr728), which is important for LF function, will be studied to provide a better understanding of the enzyme's mechanism.A second aspect of the proposed work lies in the characterization of a second zinc binding site in LF. Occupation of this site by zinc (or other metals) inhibits the enzyme, but has also been shown recently to accelerate the molecular process of exchanging one metal ion with another (e.g., replacing zinc for copper). The proposed studies will elucidate how the second metal binds to LF, and whether similar metal-binding sites found in other zinc enzymes can also facilitate metal exchange reactions. As such, our work will contribute to gaining a better understanding of the mechanism of metal exchange reactions, which are important in a number of critical biological processes.Finally, we propose to explore the possibility of using compounds which change colour upon zinc binding, as zinc accessibility probes (ZAPs) in structural investigations on LF and other zinc enzymes. Insights gained from these studies could lead to the development of new tools to probe structural aspects of metalloproteins.In summary, the proposed research will further our understanding of the role of metals in the mechanism of LF and other zinc enzymes. Such insights may be exploited for the development of novel therapeutics against LF and other structurally related zinc enzymes such as the botulinum and tetanus neurotoxins.