The overall goal of this research is to define the mechanism and role of the enzyme acetate kinase. Acetate kinase catalyzes the interconversion of acetate and acetylCoA. As acetylCoA is a key metabolite at the crossroads of many metabolic pathways, understanding acetate kinase structure, function and regulation is key to understanding basic metabolism. Although previously thought to be present primarily in bacteria, acetate kinase has now been identified in eukaryotic microbes such as fungi and protozoans. Initial characterization of acetate kinases from these organisms has revealed unique properties, suggesting that the acetate kinase enzyme family is far more diverse than previously thought. The biochemical differences between bacterial and eukaryotic acetate kinases suggest that this enzyme may serve a different physiological function in eukaryotes. This is further evidenced by the fact that phosphotransacetylase, an enzyme that functions with acetate kinase in bacteria, is absent in fungi and protozoans. The specific aims of this project are to understand the basis for the biochemical differences among the eukaryotic and bacterial acetate kinases and to determine the roles of this enzyme in fungi and protozoans. This will be the first investigation of eukaryotic acetate kinases and should shed light on additional physiological roles for this enzyme and provide unique insights into the biochemical mechanism of this important metabolic enzyme. Furthermore, acetate kinase is part of a wide family of related enzymes, which includes hexokinase, other sugar kinases, actin and Hsp70 heat shock proteins; these studies will thus provide insight into evolution of this enzyme superfamily.
Broader Impacts: The project has a broad educational impact at the graduate, undergraduate, and high school levels. The research provides a wealth of projects for students at all levels of education and ability. Projects will afford training in biochemistry, molecular biology, and microbial cell biology. Graduate students will play an essential role and will each direct an undergraduate student as well, providing an important mentoring opportunity. The research will involve high school students from the Summer Program for Research Interns at Clemson University. In addition to providing a number of graduate and undergraduate research projects at both Clemson University and Western Carolina University, this joint project also offers the Western Carolina students greater exposure to a Ph.D.-granting research university. Students involved in this project will present their research at state and regional scientific meetings as well as national meetings.
Acetate is a major metabolic product for most microbes, both prokaryotic and eukaryotic. One key route for acetate production is through the enzyme acetate kinase (ACK). This enzyme typically acts as part of a pathway, either with phosphotransacetylase (PTA) or xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (XFP). The ACK-PTA pathway is found in most bacteria although the ACK-XFP pathway is found in certain types. ACK is not widespread in eukaryotic cells, but is found in certain fungi and a few other organisms. This award was aimed at understanding the differences between prokaryotic and eukaryotic ACKs and their physiological roles, but expanded to include an investigation of the partner enzymes. Our studies indicate that differences in ACKs are not due to whether they come from prokaryotes or eukaryotes, but rather by what the partner enzyme is. ACKs that partner with PTA tend to utilize ATP, the primary energy currency of the cell, as well as other substrates closely related to ATP. ACKs that partner with XFP tend to be very narrow in their substrate range, using just ATP. We have characterized one ACK from an amoeba that does not use ATP but instead has a very different substrate preference. The partner enzyme for this ACK remains unknown, and only one instance of this unusual ACK form has been identified. We have found that in eukaryotes, the partner enzymes XFP and PTA are highly regulated, suggesting that these metabolic pathways are important and need to be controlled. A total of 7 graduate students and approximately 20 undergraduates have participated in aspects of this project. Three undergraduates were supported to continue their research full time during the summer. In addition, several students participating in a summer program for incoming honors students also participated in this project. All of these undergraduate researchers were in the lab for at least one year, with several staying for their entire undergraduate career. These students learned a wide variety of techniques beyond what they gained in their laboratory courses. The majority of these students who have graduated continued their education in Ph.D. graduate programs or medical school. This project has thus far produced four publications, with another five manuscripts submitted or soon to be submitted. In addition, our results have been well disseminated to the scientific community through presentations at research conferences, including 3 oral presentations and approximately 15 poster presentations.
