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The Paul Berg Papers

From Classical Biochemistry to Molecular Biology, 1952-1959

[Paul Berg in classroom]. [ca. 1975].
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When he chose biochemistry as his field, Berg's original plan was to pursue a career in the pharmaceutical industry. During his last undergraduate years, however, he became intrigued with recent research that used radioactive isotopes--newly available in the wake of intensive research and production during World War II--to trace the chemical changes in metabolic processes. For example, by replacing some of its carbon atoms with a radioactive carbon isotope, it was possible to track a carbohydrate's conversion from foodstuff to cellular material. These innovative methods promised to resolve many major questions about biochemical processes. Some of the most interesting work in this area was being done in the biochemistry department at Western Reserve University (now Case Western Reserve University) in Cleveland, Ohio, under the direction of Harland O. Wood, where Berg chose to do his graduate work.

In his doctoral research, Berg explored the question of how animals might synthesize methionine, an amino acid. Vincent du Vigneaud, an eminent biochemist at Cornell University, had reported that methionine was essential in animal diets, i.e., animals could not synthesize it themselves. But other investigators had found cases in which animals did well without dietary methionine, if their diets included vitamin B-12 and folic acid. One of Wood's group, Warwick Sakami, had shown (using radioisotopes) that lab rats could make the methyl groups needed for methionine and choline synthesis from injected formaldehyde. Berg's work used radioactive tracers to demonstrate in vitro that B-12 and folic acid were cofactors that facilitated the conversion of single-carbon compounds such as formic acid, formaldehyde, and methanol to methyl groups used to synthesize methionine. (Though Berg had proved him wrong about the methionine question, du Vigneaud was impressed enough to tell Wood he wanted to hire Berg, not realizing that he was still a graduate student.)

By the time his PhD was completed in 1952, Berg was committed to pursuing an academic research career rather than one in industry. His thesis project made him realize he needed further training in enzymology. Wood suggested he work in Carl Cori's lab at Washington University in St. Louis, but Berg declined to do this, partly because he'd heard about the legendary stifling summer climate there. He also wanted to work with two rising stars in enzyme chemistry whom he had met during their visits to WRU: Hermann Kalckar and Arthur Kornberg. With a postdoctoral fellowship from the American Cancer Society, he was able to arrange a year with each of them.

During 1952-53, at Kalckar's lab at the Institute of Cytophysiology in Copenhagen, Berg worked with another postdoctoral fellow, Wolfgang "Bill" Joklik, testing a colleague's proposition (based on what was known about glucose metabolism) that phosphate groups might be transferred from adenosine triphosphate (ATP--the primary energy-carrying molecule in biological systems) to a similar molecule, inosine triphosphate (ITP), using glucose and hexokinase (an enzyme that phosphorylates glucose). The process was of interest because although biochemists suspected that nucleoside triphosphates other than ATP might be energy carriers, no one had yet isolated them from living systems. Berg and Joklik tried a glucose-dependent transfer of phosphate from ATP to ITP and vice versa. They found that the phosphates could be transferred, but glucose and hexokinase have no role in the reaction. Instead, a previously unknown enzyme--which they called nucleoside diphosphokinase--uses ATP to phosphorylate the diphosphates of ribo- and deoxyribonucleosides to the respective triphosphates (the building blocks of RNA and DNA, respectively). This discovery supported earlier suggestions that other energy-carrying nucleoside triphosphate forms besides ATP could be present in biological systems.

While in Copenhagen, Berg also became intrigued by reports that Fritz Lippmann and Feodor Lynen, working independently, had solved the mystery of how eukaryotic organisms synthesize acetyl-substituted Co-enzyme A (acetyl-SCoA), a crucial intermediate in the process of food metabolism. In contrast to prokaryotes (e.g., bacteria) where synthesis occurred via two separate enzymatic reactions, Lippmann and Lynen found that yeasts and animals seemed to make it with only one enzyme, acetyl CoA synthetase, in a reaction using enzyme-bound intermediaries. They proposed the following reaction:

ATP + enzyme < - > enzyme-AMP (Adenosine monophosphate or adenylate) + PPi (inorganic pyrophosphate)

Enzyme-AMP + CoA < - > enzyme-CoA + AMP

Enzyme-CoA + acetate < - > acetyl-SCoA + enzyme

Berg wondered if such a process might also operate in the formation of similar nucleoside monophosphates besides AMP, e.g., guanosine monophosphate (GMP), cytidine monophosphate (CMP), or their deoxy- forms, which are the building blocks of RNA and DNA, respectively.

For his postdoctoral year in Kornberg's lab, Berg proposed trying to isolate this enzyme-AMP complex--Lippmann and Lynen had not isolated the enzyme, but only inferred its presence from their results. As he started to purify the enzyme, using increasingly potent solutions of it, the reaction actually stopped rather than accelerating. Puzzled, Berg broke down the reaction and tested the enzyme on each individual substrate to find out why he was getting such results from the purified enzyme. He discovered that only in the presence of acetate would the reaction proceed. Instead of Lippman and Lynen's proposed enzyme-AMP mediated reaction, the enzyme catalyzed the reaction thus:

ATP + acetate < - > acetyl AMP + pyrophosphate

Acetyl AMP + CoA < - > acetyl-SCoA + AMP

Berg's proof that acetyl AMP was the intermediate step in the formation of acetyl-SCoA caused some excitement when he reported it at the annual Federation for Experimental Biology meeting in 1955. Still just a postdoctoral fellow, he had overturned a model proposed by two senior biochemists, much to their embarrassment. In discovering the chemical pathway for acetyl-SCoA synthesis, Berg also opened the door to understanding many other biochemical reactions. It was later found that fatty acid synthesis uses a similar mechanism, and his own research soon showed that the model applied to protein synthesis as well.

At Kornberg's invitation, Berg stayed on at Washington University as a research fellow, and was appointed assistant professor in 1955. He continued to work with the synthesis model he found for acetyl-SCoA and fatty acids, and discovered enzymes that catalyze the same partial reaction with amino acids, forming enzyme-amino acyl AMP compounds. Searching for the rest of the pathway, he found that the activated amino acids bonded to a type of RNA (later called transfer RNA (tRNA) because it carries the amino acids to cell ribosomes). Berg and his first postdoctoral fellows, James Offengand and Jack Preiss, found that for each of the twenty amino acids there is a different specific enzyme that converts it to an enzyme-bound amino acyl adenylate, a step called "activation." The enzyme then transfers the amino acyl group to a specific tRNA, which takes it to ribosomes for assembly into proteins, according to the code provided by messenger RNA. Berg received the 1959 Eli Lilly Prize in Biochemistry for this work. By that time, the problems of RNA-directed protein synthesis had led him quite naturally to questions about how genes operate in the process.