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

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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.