With this collaborative RUI award, the Chemistry of Life Processes program is funding the research team of Professors Emily Fogle, Lori Robins and Kristen Meisenheimer, under the leadership of Professor John Marlier, all of Cal Poly San Luis Obispo, to study the mechanism of thioester hydrolysis. Thioesters are important intermediates in biological catalysis from fatty acid synthesis, to polyketide synthase condensations to intein self-slicing and its counterpart in modern chemical native chemical ligation. This proposal seeks to better understand the mechanism by which biologically relevant thioesters undergo hydrolysis, both in solution and in an enzyme active site. The principal tool to be employed is that of kinetic isotope effects (KIEs). Indeed, this proposal will help to bolster a standing departmental effort at Cal Poly SLO to build a thematic undergraduate research program that specializes in the techniques of KIEs to study mechanism.

The faculty team is expected to provide one-on-one training to undergraduates in modern physical organic chemistry with a focus on a biologically relevant problem. The Cal Poly team will strive to broaden participation in chemistry through the integration of women and members of traditionally underrepresented groups into the research enterprise. In the longer term, it is hoped that fundamental insights on the mechanism of thioester cleavage and condensation reactions will be obtained through these studies.

Project Report

Scientific Outcomes. The major scientific goal of this project was to investigate the chemical mechanism of thioester hydrolysis under acidic, neutral and alkaline conditions and to compare these results to those obtained for an enzyme-catalyzed hydrolysis. A chemical mechanism is a step-by-step account of what occurs between the beginning and the end of a reaction. Thioesters are a biologically important class of molecules that participate in cellular metabolism and regulation. In this project the particular thioester studied was formylthiocholine (FTC). This molecule was chosen for two reasons. First, FTC undergoes hydrolysis with and without our chosen enzyme (butyrylcholinesterase or BChE). Second, features of the molecular structure of FTC allow a broader investigation of the mechanism by two techniques: kinetic isotope effects (KIEs) and positional isotope exchange (PIX). FTC was an unknown molecule, which we synthesized and fully characterized. The structure of FTC and the equation for the hydrolysis reaction is shown in Image 1. Study of reaction mechanisms allows chemists to gain a basic understanding of short-lived, reactive intermediate chemical species that form during a reaction. In addition to increasing our basic understanding of chemical reactions, the study of reaction mechanisms often allows chemists to optimize reaction conditions. The study of enzyme-catalyzed reaction mechanisms is particularly important in biology because some studies of enzyme mechanisms have resulted in drug development. An example of a mechanism with reactive intermediates is shown in Image 2. The first type of experiment in our project followed how fast a water molecule containing a heavy isotope of oxygen (18O) exchanged into the reactant (FTC) compared to how fast the product formed. These PIX experiments can offer evidence for the existence of certain symmetric reactive intermediates in the mechanism and can also indicate how fast these intermediates form the product versus how fast they return to the reactant. An example of such a symmetrical intermediate (shown in brackets) is given in Image 3. In all reaction conditions studied PIX experiments determined that the symmetrical intermediates formed product much faster than they returned to a reactant containing an 18O label. Therefore, breaking the C—S bond is very rapid relative to the other steps in the mechanism. Unfortunately the PIX results for the enzyme-catalyzed hydrolysis are more difficult to interpret due to an inherent property of enzymes. The second type of experiment was KIEs. Here the rate of hydrolysis of FTC containing a lighter isotope in a particular position was compared to that containing a heavier isotope in the same position. These experiments are summarized in Image 4. Each type of KIE was repeated for acidic, neutral, alkaline and enzyme-catalyzed hydrolysis. The results of our KIE experiments helped determine which chemical step in the mechanism was the slowest step. Chemists call this the rate-determining step. For hydrolysis under acidic and neutral conditions the KIEs are consistent with the rate-determining step being the formation of the first reactive intermediate. In addition, the results from these KIE experiments indicate that the highest energy point during this step occurs when formation/breaking of the bonds is in its early stages. Chemists refer to these highest energy points as transition states; those that occur during the early stage of bond formation/breaking are called early transition states. Under alkaline conditions the highest energy point is also formation of the first intermediate but the results argue for more fully formed/broken bonds making this a late transition state. BChE-catalyzed hydrolysis has two distinct steps: acylation and de-acylation. This mechanism is shown in Image 5. Most of our KIE results are limited to studying only the acylation step. These results indicate that formation of the first reactive intermediate is probably rate-determining. In addition, the results indicate that the transition state would be considered early, just like in the alkaline hydrolysis case discussed above. Ongoing work on the sulfur KIE will further clarify this mechanism. In contrast to the KIE experiments discussed above, the nucleophile-O KIE only gives results on the de-acylation step. In this case the observed results argue that the formation of the first intermediate during de-acylation is rate-determining with a late transition state. Broader Outcomes. NSF support for this project offered two other broad impacts: (1) Cal Poly is an undergraduate institution. NSF funding provides stipend support for undergraduate students to conduct research on the project as training for future careers in chemistry, biochemistry and medicine. (2) The principle investigator (PI) has more than thirty years of research experience in the field and provides mentoring for the younger co-PIs, who have been formally trained in this area of research, but lack the years of experience. In the process, this management plan allowed for a well-supervised undergraduate research experience, as well as for establishment of an ongoing high-level research program in bioorganic chemistry for the faculty PI and co-PIs.

National Science Foundation (NSF)
Division of Chemistry (CHE)
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Colby A. Foss
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California Polytechnic State University Foundation
San Luis Obispo
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