History of Biology: Biochemistry

Biochemistry as a recognizably distinct discipline emerged at the beginning of the twentieth century. Initially it focused on the chemical changes of cel­lular metabolism.

Roots of Biochemistry

Biochemistry had its roots in nineteenth-century physiological chemistry, animal chemistry, and the chemistry of biological materials. The earliest

Eduard Buchner, whose experiments with zymase initiated the study of biochemistry.

views on the chemistry of life posited that it was fundamentally different from nonliving chemistry. From around 1835, the view had developed that protoplasm, seen as a jellylike single homogeneous form of matter within organisms, carried out all the processes of intracellular breakdown of foods, respiration, and biosynthesis. Despite this general belief, neither Justus Liebig nor Ernst Hoppe-Seyler, two eminent chemists, accepted this view. Hydrolytic enzymes such as amylase, maltase, and pepsin were known in the nineteenth century, but were not thought to act within cells.

Probably the single most important experiment that initiated the study of biochemistry was the preparation by Eduard Buchner in 1897 of a cell- free extract of yeast, called zymase, which fermented glucose and produced carbon dioxide and ethanol. Buchner regarded zymase as a single enzyme, although others soon showed that it contained several. This work confirmed fermentation as a chemical process and discredited the protoplasm theory. Furthermore, the distinction between catalysis by hydrolytic extracellular enzymes and by intracellular enzymes disappeared.

Enzymes

The nature of catalysis began to be explored early in the twentieth century with the realization that enzymes bind their substrates during the reaction. At the beginning of the twentieth century, Emil Fischer proposed that a substrate fits its enzyme like a key fits a lock. Mathematical analysis of en­zyme action enabled Leonor Michaelis and Maud Menten to formulate the classic equations for enzyme action in 1913. The chemical nature of en­zymes as proteins remained uncertain until James Sumner crystallized ure­ase from Jack bean meal in 1926. Several other enzymes were crystallized in the following years and were all shown to be proteins. In 1959, Sanford Moore and William Stein determined the first primary sequence (the amino acid sequence) of an enzyme, ribonuclease. The path was now open, using X-ray crystallography to reveal the catalytic process in three-dimensional models of enzymes.

Metabolism

Part of the significance of Buchner’s work lay in initiating the study of fer­mentation as a metabolic pathway. Otto Meyerhof demonstrated that mus­cle juice had similar properties, although producing lactic acid rather than ethanol. Thus, the glycolytic pathway, associated with the names of Gustav Embden, Meyerhof, and Otto Warburg, was elucidated over the first four decades of the twentieth century.

In the first years of the twentieth century, Franz Knoop and also Henry Dakin outlined the basis of fatty acid oxidation, although this pathway was not fully formulated until the 1950s. The cyclical nature of some metabolic pathways became apparent to Hans Krebs in his study of the synthesis of urea, which led to the description of the urea cycle in 1931. In 1937, build­ing on much work on cell oxidation reactions, Krebs formulated the citric acid cycle (often called the Krebs cycle in his honor). Identification of acetyl coenzyme A in the early 1950s facilitated the understanding of pyruvate ox­idation, fatty acid oxidation, and the citric acid cycle.

During the 1930s, biochemists began to use radioactive isotopes such as deuterium (²H), ³²P, and ³⁵S in studies of metabolism. After World War II, ¹⁴C became readily available, and together with the use of microbial mu­tants, enabled researchers to elucidate metabolic pathways, particularly from 1945 to 1975.

Bioenergetics and Membranes

The importance of adenosine triphosphate (ATP) emerged slowly from a study of the cofactors necessary for glycolysis in yeast and muscle. In 1939, Vladimir Engelhardt and Militsa Lyubimova showed ATP to be a substrate for myosin that participates in muscular contraction. In 1941, Fritz Lip- mann set out the essential role of ATP as the energy currency of the cell. Initially ATP synthesis was thought to be associated only with glycolysis, but during the 1930s a number of studies showed that much of ATP pro­duction was associated with oxygen uptake, linking oxygen consumption with phosphorylation.

During the 1920s and 1930s, David Keilin outlined the steps of the res­piratory chain, through which oxygen is consumed in the mitochondrion. By the 1950s, it was clear that the respiratory chain was coupled to the syn­thesis of ATP. In 1961, Peter Mitchell formulated the chemiosmotic theory of ATP production, based on a gradient of H⁺ ions and membrane potential. This not only resolved the issue of oxidative phosphorylation but also gave a firm foundation for studies of transport across membranes. Jonathon Singer and Garth Nicholson resolved problems of membrane structure by proposing in 1972 the fluid mosaic model in which proteins are embedded in a fluid lipid bilayer.

At the beginning of the twentieth century, the basic process of photo­synthesis had already been defined. From 1954 to 1956, Melvin Calvin and Andrew Benson formulated the metabolic pathway for carbon dioxide fixa­tion, which would be named the Calvin-Benson cycle in their honor. In 1937, Robin Hill and others had progressively elaborated the workings of the electron transport chain of the chloroplast. In the second half of the 1950s, Daniel Arnon showed that the electron transport chain drives ATP synthesis in the chloroplast. Many researchers in the twentieth century ex­plored the basic photochemistry of photosynthesis, but the determination of the structure of a bacterial reaction center complex by Johann Deisen- hofer in 1984 has helped to clarify the field.

Proteins

The theory that proteins were composed of linear chains of amino acids had been enunciated at the beginning of the twentieth century, while the amino acid constituents of proteins were still being identified. However, it was not until Fred Sanger obtained the first complete primary structure of a protein, insulin, in 1955 that the theory was confirmed. A model of the three-dimensional structure of a protein (whale myoglobin) was de­termined by John Kendrew and colleagues between 1958 and 1960 based on X-ray analysis. Concurrently Max Perutz described the structure of hemoglobin. Among other issues, these achievements confirmed the pre­dictions made in 1950 by Linus Pauling and Robert Corey that the amino acid chain could coil into a spiral staircase-like structure called the alpha helix. Researchers have described the three-dimensional structures of many proteins since that time.

Improved techniques of crystallography, X-ray analysis, and other skills in protein chemistry are bringing new insights to many areas of classical biochemistry, including enzymology. For example, the elucidation of the structure and mechanism of the bacterial mitochondrial and chloroplast ATP synthase (ATPase) in the 1990s, based on the work of Paul Boyer and John Walker, has revealed an enzyme that involves a rotating core to carry out ATP synthesis. This molecule is used by both mitochondria and chloro- plasts to make ATP.

DNA and Protein Synthesis

Work on bacteria led Oswald Avery to suggest in the 1940s that deoxyri­bonucleic acid (DNA) was the genetic material of the cell. Francis Crick and James Watson elucidated the structure of DNA in 1953. The structure they proposed suggested immediately the way in which genes might be repli­cated. More importantly, Crick suggested that the sequence of bases deter­mines the sequence of amino acids in proteins, and that the amino acid sequence in itself determined the three-dimensional structure of these pro­teins. By 1960, it was believed that a sequence of three DNA bases encodes an individual amino acid. Marshall Nirenberg, Severo Ochoa, and Gobind Khorana “cracked” the genetic code—the specific DNA base triplets that specify particular amino acids—in a series of experiments conducted in the 1960s.

In 1961, Francois Jacob and Jacques Monod introduced the concept of regulatory genes, which control the expression of structural genes that en­code for proteins. Other methods of control were discovered later. By the 1970s, it was possible to synthesize proteins in vitro.

Manipulation of DNA and Elucidation of Protein Structure

The sequencing of significant lengths of DNA remained a problem until in 1977 Fred Sanger sequenced a bacteriophage genome of 5,375 nucleotides. This achievement led the way to Sanger’s sequencing of the DNA of the human mitochondrial genome of more than 16,000 nucleotides in 1981. These early sequencing successes contributed to the idea that arose in the mid-1980s to sequence the human genome, which researchers expect to complete in 2003 (a draft sequence was published in 2001).

Parallel to DNA sequencing efforts was the use of restriction enzymes, which cut DNA at specific sequences. Restriction enzymes made possible DNA sequencing, recombinant DNA technology, transgenic technology, and other tools, giving rise to the rapid development in the 1990s of biotech­nology based on gene splicing.

References

Crick, Francis. What Mad Pursuit: A Personal View of Scientific Discovery. New York: Basic Books, 1988.

Florkin, Marcel, and Stotz, Elmer H. Comprehensive Biochemistry, Section VI (Vol­umes 30-39): A History of Biochemistry. Amsterdam: Elsevier, 1970-95.

Fruton, Joseph S. Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology. New Haven, CT: Yale University Press, 1999.

Judson, Horace F. The Eighth Day of Creation. New York: Simon & Schuster, 1979.

Kohler, Robert E. “The Enzyme Theory and the Origin of Biochemistry.” Isis 64 (1973): 181-196.

Krebs, Hans A. Reminiscences and Reflections. Oxford: Oxford University Press, 1981. Watson, James D. The Double Helix. London: Weidenfeld & Nicolson, 1968.