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Fatty Acid Synthesis and Glutamine Synthetase: the Work of Earl Stadtman

2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês

10.1016/s0021-9258(20)65687-3

1083-351X

Nicole Kresge, Robert Simoni, Robert L. Hill,

Advanced Proteomics Techniques and Applications

Fatty Acid Synthesis by Enzyme Preparations of Clostridium kluyveri. I. Preparation of Cell-free Extracts That Catalyze the Conversion of Ethanol and Acetate to Butyrate and Caproate (Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093) Allosteric Regulation of the State of Adenylylation of Glutamine Synthetase in Permeabilized Cell Preparations of Escherichia coli. Studies of Monocyclic and Bicyclic Interconvertible Enzyme Cascades, in Situ (Mura, U., Chock, P. B., and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029) Earl Reece Stadtman was born in 1919 in Carrizozo, a small town in New Mexico. When he was 10, his family moved to San Bernardino, California, where he attended high school. After graduating from high school in 1937, Stadtman enrolled in several science courses at San Bernardino Valley College, hoping to eventually set up a soil-testing laboratory. However, he soon realized that he needed a more rigorous education and enrolled at the University of California, Berkeley. He earned a B.S. in soil science in 1942. After spending a year in Alaska, involved in a wartime project of mapping the Alaskan-Canadian (Al-Can) Highway, Stadtman returned to Berkeley looking for work. He paid a visit to Horace A. Barker, a Berkeley biochemist for whom he had worked as a laboratory technician (and author of a future Journal of Biological Chemistry (JBC) Classic). At that time, Barker was directing various war efforts in the Department of Food Technology and offered Stadtman a job as principal investigator on a project studying the “Browning of Dried Apricots,” the goal of which was to find a way to slow the deterioration of dried fruits during storage. Around this time, Stadtman also met his future wife, Thressa Campbell, who was working as a laboratory assistant in the food technology department. After the war, Stadtman started graduate studies in the Department of Biochemistry working in Barker's laboratory. Barker had spent a year as a postdoc in Albert J. Kluyver's laboratory at the Technical School in Delft in the Netherlands before coming to Berkeley and had isolated a species of bacteria called Clostridium kluyveri (named after Kluyver) from the Delft canal mud. Since then, Barker had been searching for an explanation for the observation that C. kluyveri could produce short-chain fatty acids from ethyl alcohol. He made a breakthrough when he obtained some 14C and used the isotope to label acetate and demonstrate that fatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules (1Barker H.A. Kamen M.D. Bornstein B.T. The synthesis of butyric and caproic acids from ethanol and acetic acid by Clostridium kluyveri..Proc. Natl. Acad. Sci. U. S. A. 1945; 31: 373-381Crossref PubMed Scopus (57) Google Scholar). He deduced that ethanol is first oxidized to “active” acetate (a 2-carbon compound), which is condensed with acetate to form a 4-carbon compound that is reduced to form butyrate. Active acetate can then be condensed with butyrate to form caproate. Barker surmised that acetyl phosphate might be the active acetate formed in the above reaction. It was at this point that Stadtman joined Barker's laboratory and started working on fatty acid synthesis. Initially, like Barker, Stadtman used 14C to trace the metabolic pathways in whole cell preparations of C. kluyveri. However, he abandoned this approach after a visit to Irwin C. Gunsalus's laboratory at Cornell University. Gunsalus showed Stadtman how to dry bacterial cells and grind the dried preparations to break open cell walls, producing a cell-free extract. Applying this method to C. kluyveri, Stadtman was able to produce extracts that could catalyze all of the reactions involved in the conversion of ethanol and acetate to fatty acids of 4- and 6-carbon atoms. His preparations also catalyzed the aerobic oxidation of ethanol and butyrate. These experiments are reported in the first JBC Classic reprinted here. This discovery was especially significant because up until that time most biochemists believed that the capacity to make fatty acids was a unique property of specialized cellular systems or particulate organelles. In a series of additional papers (2Stadtman E.R. Barker H.A. Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. II. The aerobic oxidation of ethanol and butyrate with the formation of acetyl phosphate..J. Biol. Chem. 1949; 180: 1095-1115Abstract Full Text PDF PubMed Google Scholar, 3Stadtman E.R. Barker H.A. Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. III. The activation of molecular hydrogen and the conversion of acetyl phosphate and acetate to butyrate..J. Biol. Chem. 1949; 180: 1117-1124Abstract Full Text PDF PubMed Google Scholar, 4Stadtman E.R. Barker H.A. Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. IV. The phosphoroclastic decomposition of acetoacetate to acetyl phosphate and acetate..J. Biol. Chem. 1949; 180: 1169-1186Abstract Full Text PDF PubMed Google Scholar, 5Stadtman E.R. Barker H.A. Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. V. A consideration of postulated 4-carbon intermediates in butyrate synthesis..J. Biol. Chem. 1949; 181: 221-235Abstract Full Text PDF PubMed Google Scholar, 6Stadtman E.R. Barker H.A. Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. VI. Reactions of acyl phosphates..J. Biol. Chem. 1950; 184: 769-794Abstract Full Text PDF PubMed Google Scholar), all published in the JBC, Stadtman and Barker used the enzyme extracts to study the individual reactions involved in fatty acid synthesis and confirmed that ethanol is oxidized to acetyl phosphate, which condenses with acetate and forms butyric acid. They also discovered that C. kluyveri contained an acetyl-transferring enzyme (phosphotransacetylase) and an enzymatic system for using acetyl phosphate to activate other fatty acids. Stadtman later showed that acetyl-CoA was the source of active acetate in the synthesis of butyric acid from acetyl phosphate (7Stadtman E.R. Novelli G.D. Lipmann F. Coenzyme A function in and acetyl transfer by the phosphotransacetylase system..J. Biol. Chem. 1951; 191: 365-376Abstract Full Text PDF PubMed Google Scholar) while working as postdoctoral fellow with Fritz Lipmann (author of a previous JBC Classic (8JBC Classic Lipmann F. J. Biol. Chem. 1945; 160 (http://www.jbc.org/cgi/content/full/280/21/e18): 173-190Abstract Full Text PDF Google Scholar)). In 1950, Stadtman began to look for an academic position. However, because his wife Thressa also had a Ph.D., they were looking for an institution at which they could both work at the same professional level. Unfortunately, at that time, most universities had anti-nepotism rules that did not allow more than one family member to work in the same department. Intended to protect universities from charges of favoritism, the rules often had the effect of discriminating against married women. No one seriously challenged the rules until the 1960s, when the American Association of University Women began to protest their unfairness. Fortunately, these polices were not in effect at the National Institutes of Health (NIH), and in September 1950, the Stadtmans moved to Bethesda, Maryland. Both continue to do research at the NIH today. At the NIH, Stadtman continued his research on fatty acid metabolism. In 1952, he successfully carried out the first in vitro net synthesis of acetyl-CoA using only basic materials (acetyl phosphate, CoA, and phosphotransacetylase). Stadtman and his postdoc P. Roy Vagelos also demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is the source of active acetate. Another topic of long term research in Stadtman's laboratory was glutamine synthetase, the enzyme that catalyzes the conversion of glutamate to glutamine. The activity of glutamine synthetase is subject to feedback inhibition by 7 different end products of glutamine metabolism. Stadtman discovered that this end product inhibition was cumulative (the presence of more end products resulted in more inhibition) and that susceptibility to feedback inhibition only occurred when glutamine synthetase was adenylated by adenylyltransferase (ATase). He later found that adenylation was regulated by uridylyltransferase (UTase), which, depending on the cellular concentration of various metabolites, catalyzed the covalent attachment of a uridylyl group to the regulatory protein, PII. The uridylated form of PII stimulates ATase to catalyze glutamine synthetase deadenylation, whereas the unmodified form of PII stimulates ATase catalysis of the adenylation reaction. In view of these results, Stadtman surmised that glutamine synthetase activity was controlled by a cascade system in which two systems of reversible covalent modification were tightly linked. Each system was composed of two reversible reactions, or two interconvertible enzyme cycles, the linkage of which resulted in the formation of a bicyclic cascade system. This cascade system allowed enzyme activity to be shifted gradually in response to metabolite availability. In the late 1970s and early 1980s, Stadtman and P. Boon Chock carried out a theoretical analysis of this bicyclic cascade system to understand its implications in enzyme regulation. However, it was not until 1981 that they were able to study the cascade in vivo. These experiments are discussed in the second JBC Classic reprinted here. Stadtman had discovered that after a freeze-thaw cycle, treatment of Escherichia coli cells with a nonionic detergent rendered them permeable to small metabolites but allowed the cells to retain the protein components of the cascade system. Furthermore, permeabilized cells from cultures containing 10 mm glutamine retained all their cascade enzymes whereas 5 mm glutamine-grown cells had inactivated UTase. Using these cells, they were able to study the effects of different substrates and allosteric effects on the cascade system and to confirm the previous theoretical and in vitro studies. A more complete description of Stadtman's work on glutamine synthetase can be found in his JBC Reflections (9Stadtman E.R. The story of glutamine synthetase regulation..J. Biol. Chem. 2001; 276: 44357-44364Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Stadtman has received many awards and honors for his numerous research discoveries including the 1979 National Medal of Science, the 1983 ASBC-Merck Award, and the 1991 Robert A. Welch Award in Chemistry. Stadtman was also President of the American Society for Biological Chemists (now American Society for Biochemistry and Molecular Biology) from 1982 to 1983 and has been a member of the National Academy of Sciences since 1969. 1All biographical information on Earl R. Stadtman was taken from Ref. 10, Park, B. S. The Stadtman way: A tale of two biochemists at NIH. http://history.nih.gov/exhibits/stadtman/index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart, Lung, and Blood Institute)Google Scholar. 1All biographical information on Earl R. Stadtman was taken from Ref. 10, Park, B. S. The Stadtman way: A tale of two biochemists at NIH. http://history.nih.gov/exhibits/stadtman/index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart, Lung, and Blood Institute)Google Scholar.