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The Arthur Kornberg Papers

From Physician to Enzyme Hunter, 1942-1953

[Arthur Kornberg at NIH with DeWitt Stetten]. 13 October 1959.
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When ship's doctor Arthur Kornberg was reassigned to a research post at the National Institute of Health (NIH)--now the National Institutes of Health--in 1942, he did not expect to stay there beyond the end of World War II. He had no formal research qualifications, apart from his small medical school study of benign jaundice. Neither his medical education nor his internship had included any research training. Within a few short years, however, he had found his calling as a biochemist specializing in enzymology, become the director of an enzyme laboratory at NIH, and discovered how several key metabolic enzymes are made.

Kornberg began his NIH work at the Nutrition Laboratory, where the research focused on finding vitamins and other essential nutrients, and on investigating various aspects of deficiency diseases in animals and humans. Many vitamins had been discovered between 1880 and 1940, solving the mysteries of deficiency diseases such as beri-beri, pellagra, scurvy, and rickets. But much remained unknown about how they functioned in the body, and about how other chemicals might aid or hinder their actions. Kornberg's first research project was to study the effects of sulfadiazine and sulfathiazole (recently-developed antibiotic sulfa drugs) on rats fed on a purified nutrient diet. Rats on such a diet (glucose, milk protein, cod liver and cottonseed oils, and known vitamins) stayed healthy until they were given sulfa. Within a few weeks, many of them developed fatal blood disorders. If, however, they were fed regular animal rations or a liver supplement, the blood disorders could be prevented or cured. The curative ingredient in liver, found also in yeast and certain vegetables, proved to be folic acid. Kornberg and his colleagues discovered that sulfa drugs are structurally quite similar to para-aminobenzoic acid (PABA), a key component of folic acid. Sulfa competes with PABA for the enzyme that assembles folic acid, and so prevents its manufacture by intestinal bacteria. (Animals on the synthetic diet, with no supplemental folic acid, could get by on the amounts produced by their intestinal bacteria, until the sulfa drugs were given.) Kornberg did a related study on the link between sulfa drugs and vitamin K deficiency, finding that, again, the sulfa drugs prevented the intestinal bacteria from manufacturing folic acid, thus killing them. Because the bacteria also make vitamin K (the substance essential for blood clotting), sulfa drugs also induced a deficiency of that nutrient.

By 1945, Kornberg was becoming much more interested in how vitamins work than in discovering new ones. Many vitamins function as components of enzymes, the large specialized proteins that drive all body processes, by assembling or breaking down larger molecules. His nutritional work had led to an interest in the metabolic enzymes, which catalyze the breakdown of glucose (sugar) to generate energy for growth and work in all living systems. By 1945, chemists had discovered the outline of the basic processes of glucose metabolism, in which glucose was converted to pyruvic acid and then to energy (in the form of adenosine triphosphate--ATP) via the citric acid cycle worked out by Hans Krebs in 1937. But the details of ATP production--including many enzymes--were still unknown. As a first step, Kornberg apprenticed himself to Bernard Horecker, a colleague at the NIH Industrial Hygiene Research Division, who had some experience with metabolic enzyme research. Together they explored one step of the citric acid cycle involving succinic acid. They isolated the associated enzyme (succinic acid oxidase) and monitored the transformation of succinic acid through several steps. Their investigations failed to reveal the major source of ATP production; their enzyme extract would catalyze the reaction, but was too crude to allow a detailed analysis. It became clear to Kornberg that he would need to master the art of enzyme purification to pursue his interest in the ATP production process.

Kornberg persuaded his division chief, Henry Sebrell, to grant him leave to get further training in enzyme chemistry. He went first to work with Severo Ochoa at New York University, who was purifying enzymes likely to be involved in ATP synthesis (also called oxidative phosphorylation.) Ochoa assigned him to purify the enzyme aconitase from pig hearts and pigeon breasts, and he labored at this for six months, learning the "philosophy and practice of enzyme purification." Cells contain thousands of different enzymes plus many other proteins. To study a particular enzyme, it must be separated from all the other cell proteins, as their presence will interfere with the desired reactions and measurements. The targeted enzyme from animal tissue or a culture of bacterial cells is separated from the other cell proteins by various chemical and physical means. At each step, the investigator tests the activity of the enzyme fraction on its substrate (the specific material on which a given enzyme will catalyze a reaction). When very small amounts of enzyme solution can induce a large reaction, the sample is considered fairly pure. Kornberg spent all of 1946 learning these painstaking techniques from Ochoa, supplementing the lab training with summer chemistry courses at Columbia University.

In January 1947, he moved to Washington University in St. Louis to work with Carl and Gerty Cori (who would receive the Nobel Prize later that year for their work on glucose metabolism). In the Cori lab, his research problem was to account for the presence of inorganic pyrophosphate when liver tissue metabolized pyruvic acid. The pyrophosphate was previously unknown as a cell constituent, but possessed a chemical energy similar to that of ATP. Although Kornberg did not discover much about pyrophosphate, he did find that the cellular respiration process was strongly enhanced by the coenzyme NAD (nicotinamide adenine dinucleotide), which was split by another enzyme, liberating AMP (adenosine monophosphate) to stimulate the reaction. (Coenzymes are non-protein components of enzymes, essential for their catalytic action; many vitamins function as coenzymes in the body.)

When Kornberg returned to NIH in the fall of 1947 to organize an Enzyme Section within the Institute of Arthritis and Metabolic Diseases, he abandoned his search for the source of ATP (it would later turn out that the enzymes for this synthesis don't exist in discrete soluble forms, but are embedded in the walls of cell structures called mitochondria). Instead he decided to find out more about the enzyme that split NAD. He was able to extract the enzyme (nucleotide pyrophosphatase) easily from potatoes. Soon he discovered that it would split not only NAD but many similar compounds. Having found the enzyme that splits NAD, he wondered if one existed that assembles it. He found that there was--NAD synthetase. Once he had isolated this he was able to delineate the reaction, and found that it produced pyrophosphate along with NAD, which explained how the former ended up in the liver tissue he had studied in the Cori lab.

The synthesis of NAD suggested that a similar mechanism might be involved in making other metabolic coenzymes such as FAD (flavin adenine dinucleotide), and Kornberg subsequently found enzymes that synthesized several of them. This work produced four scientific papers and earned him the Paul-Lewis Award in enzymology (now the Pfizer Award) in 1951.

These were key discoveries: the basic synthesis mechanism that Kornberg identified for these coenzymes was found to operate in the synthesis of many other large molecules. In each case, building blocks of proteins, carbohydrates, lipids, or nucleic acids, with the right enzyme, will react with ATP or related substances, release pyrophosphate, and bond together in the characteristic large molecule form.