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Chester H. Werkman and Merton F. Utter: Using Bacteria Juice and 13C to Explore Carbon Dioxide Fixation

2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês

10.1016/s0021-9258(20)69340-1

1083-351X

Nicole Kresge, Robert Simoni, Robert L. Hill,

Microbial metabolism and enzyme function

Reversibility of the Phosphoroclastic Split of Pyruvate (Utter, M. F., Lipmann, F., and Werkman, C. H. (1945) J. Biol. Chem. 158, 521–531) Chester Hamlin Werkman (1893–1962) was born in Fort Wayne, Indiana. His career in science began at Iowa State University in 1920 when he became a graduate student under Robert E. Buchanan, an internationally recognized microbiologist. Werkman's interest in bacteria stemmed from the fact that he thought of them as simple models for studying the basic chemical transformations involved in living processes. He completed his dissertation in 1923 and remained with Buchanan until he was offered a faculty position at the University of Massachusetts in 1924. However, he returned to the Department of Bacteriology at Iowa State a year later and remained there as a faculty member for the rest of his life. Upon returning to Iowa State, Werkman's research interests underwent a slow evolution. Initially he continued to publish papers related to his thesis work on immunology and vitamins, but soon he developed an interest in food microbiology and the role of vitamins as growth factors for bacteria. During the early 1930s, the Iowa State agricultural experimental station started investigating the use of bacterial fermentation to dispose of farm waste, and Werkman became involved in this effort. This resulted in his publication of a series of papers describing organic techniques to isolate and quantify the products of various fermentation processes. Soon Werkman became interested in investigating the intermediate mechanisms of these fermentations and embarked on what would become a lifelong study of reaction intermediates in bacteria. One of Werkman's most important contributions to physiological microbiology was done with his graduate student, Harland G. Wood. Werkman and Wood established the existence of heterotrophic carbon dioxide fixation (the concept that all organisms, not just plants or specialized bacteria, can utilize CO2), which could be summarized by the “Wood Werkman reaction.” CO2+CH3COCOOH⇌COOHCH2COCOOH Werkman and Wood used 13C-labeled compounds to confirm heterotrophic carbon dioxide fixation and also to study the utilization of carbon in metabolism. To do this, they built a mass spectrometer and a 72-foot thermal diffusion column (to produce concentrated 13C) in the elevator shaft of the science building. They published their first detailed papers on the use of 13C-labeled compounds in the Journal of Biological Chemistry (JBC) (1Wood H.G. Werkman C.H. Hemingway A. Nier A.O. Heavy carbon as a tracer in heterotrophic carbon dioxide assimilation..J. Biol. Chem. 1941; 139: 365-376Abstract Full Text PDF Google Scholar, 2Wood H.G. Werkman C.H. Hemingway A. Nier A.O. The position of carbon dioxide carbon in succinic acid synthesized by heterotrophic bacteria..J. Biol. Chem. 1941; 139: 377-381Abstract Full Text PDF Google Scholar). These and other papers by Wood will be the subject of a future JBC Classic. In 1938, Merton Franklin Utter (1917–1980) joined Werkman's laboratory as a graduate student. Utter, who was born in Westboro, Missouri, had just graduated from Simpson College in Indianola, Iowa. The first paper he published with Werkman was entitled “The Preparation of an Active Juice from Bacteria” (3Wiggert W.P. Silverman M. Utter M.F. Werkman C.H. Preparation of an active juice from bacteria..Iowa State Coll. J. Sci. 1940; 14: 179-186Google Scholar). This was a very modest title considering that active enzyme systems had not yet been isolated from bacteria. Werkman and Utter used these bacterial extracts and 13C-labeled compounds to further investigate carbon dioxide fixation, which is the subject of the JBC Classic reprinted here. The Wood Werkman reaction had already established that carbon dioxide could be combined with aC3 compound, but the existence of a C1 + C2 reaction had not been demonstrated. Werkman and Utter knew that the phosphoroclastic split, in which pyruvic acid is split to make acetyl phosphate and formic acid, was common in Escherichia coli. CH3COCOOHPyruvic acid+H3PO4⇌CH3COOPO3H2acetyl phosphate+HCOOHformic acid If they could prove that this reaction was reversible, it would be an example of a C1 + C2 addition. Although the C1 compound in the reaction is formic acid rather than carbon dioxide, formic acid is in equilibrium with carbon dioxide and hydrogen in E. coli, so carbon dioxide fixation is ultimately involved in the reaction. Werkman and Utter teamed up with Fritz Lipmann (the author of a future JBC Classic), who had just discovered the role of acetyl phosphate in metabolism. To prove the reversibility of the reaction, they added 13C-labeled formic acid to E. coli extracts and tested for 13C in the resulting pyruvic acid. In separate experiments they added CH 133COOH and adenyl pyrophosphate (which would react to form labeled acetyl phosphate) to the extracts. In both cases the pyruvic acid formed contained 13C, demonstrating the reversibility of the phosphoroclastic split and the occurrence of C1 + C2 carbon dioxide fixation. As a final test they added 13CO2 to whole cell suspensions of E. coli and showed that the bacteria produced 13C-labeled pyruvic acid. After earning his Ph.D. with Werkman in 1942, Utter was appointed instructor in bacteriology at Ohio State. In 1944 he was offered an assistant professorship at the University of Minnesota and moved to Minneapolis. He moved again in 1946, this time to Cleveland, Ohio, to become an associate professor of biochemistry at Western Reserve University School of Medicine. Utter was promoted to professor in 1956 and became chairman of the biochemistry department in 1965. He remained as chairman until 1976 and then devoted all of his time to research and teaching in the Department of Biochemistry. Utter was also an associate editor for the JBC and helped to guide the journal's editorial policies during its rapid expansion. Utter continued to study metabolism and soon became interested in gluconeogenesis, which is where he made his most significant contribution to biochemistry. For many years it was believed that the synthesis of glucose (gluconeogenesis) occurred by the reversal of the Embden-Meyerhof pathway in glycolysis. Utter demonstrated that this was incorrect by discovering phosphoenolpyruvate carboxykinase and pyruvate carboxylase, two enzymes that are involved in the conversion of pyruvate to phosphoenolpyruvate in a sequence of reactions that differ from those in glycolysis. Utter, along with Bruce Keech, also provided one of the first examples of allosteric control of an enzyme when he demonstrated that acetyl-CoA regulates pyruvate carboxylase activity. 1All biographical information on Chester Hamlin Werkman was taken from Refs. 4Brown R.W. Biographical Memoir of Chester Hamlin Werkman. 44. National Academy of Sciences, Washington, D. C.1974: 328-358Google Scholar and 5Singleton R. From bacteriology to biochemistry: Albert Jan Kluyver and Chester Werkman at Iowa State..J. Hist. Biol. 2000; 33: 141-180Crossref PubMed Scopus (10) Google Scholar. 1All biographical information on Chester Hamlin Werkman was taken from Refs. 4Brown R.W. Biographical Memoir of Chester Hamlin Werkman. 44. National Academy of Sciences, Washington, D. C.1974: 328-358Google Scholar and 5Singleton R. From bacteriology to biochemistry: Albert Jan Kluyver and Chester Werkman at Iowa State..J. Hist. Biol. 2000; 33: 141-180Crossref PubMed Scopus (10) Google Scholar., 2All biographical information on Merton Franklin Utter was taken from Ref. 6Wood H.G. Hanson R.W. Biographical Memoir of Merton Franklin Utter. 56. National Academy of Sciences, Washington, D. C.1987: 474-499Google Scholar. 2All biographical information on Merton Franklin Utter was taken from Ref. 6Wood H.G. Hanson R.W. Biographical Memoir of Merton Franklin Utter. 56. National Academy of Sciences, Washington, D. C.1987: 474-499Google Scholar.