Biochemistry

Definition: Biochemistry involves the study of chemical processes in cells and organisms, which may include metabolic pathways and the regulation of such pathways. Biochemistry may also focus on the study of enzymes that catalyze metabolic pathways. Because cell metabolism utilizes a complex interaction of enzymes and the molecules they act upon (called substrates), an understanding of metabolism is key to defining the cell itself. Disruption of metabolic pathways may result in metabolic diseases, either hereditary (genetic) changes or alterations that result from exposure to environmental factors. Since at the molecular level, cancer is the result of genetic and biochemical changes that take place in the cell, oncology can include the application of biochemistry in understanding the significance of these changes.

Basic Principles

While biochemistry encompasses the study of any cell process using either organic (carbon-carbon bonding) or inorganic substances, its eighteenth-century origins focused largely on the chemistry of plant and animal proteins. Scientists recognized that protein could be digested using stomach secretions and, later, observed that saliva would convert starch into simpler sugars. However, the mechanism behind these reactions remained a mystery. In the nineteenth century, French chemist Louis Pasteur hypothesized that the process of sugar converting to alcohol during fermentation must be catalyzed (sped up) by a substance synthesized in living cells. Some years later, the term “enzyme” was coined by German physiologist Wilhelm Kühne to explain the reaction. During the 1920s, James Sumner demonstrated the protein nature of most enzymes.

Biochemistry is most widely used in the field of cell metabolism, encompassing any chemical reactions that take place in cells. The concept includes a wide range of chemical applications of events in the cell, from regulation of gene expression to regulation of metabolic pathways. The biochemistry of cells is often likened or simplified to a series of chemical reactions that either synthesize metabolic compounds (anabolism) or break down metabolic compounds (catabolism). The processes may release energy, usually in the form of the molecule adenosine triphosphate (ATP), or utilize energy, commonly the breakdown of ATP, in the reaction. The series of reactions constitutes the metabolic pathway.

Metabolic pathways commonly begin with a simple sugar such as glucose, the complete oxidation of which produces water and carbon dioxide in a process called respiration. Depending on the type of cell, many of which carry out pathways unique to a specific organism, only portions of a pathway may be important. However, descriptions of the biochemical pathways of metabolism remain relevant because, regardless of the starting or stopping points of the metabolic reactions in cells, the steps commonly overlap with the respiration pathway.

Core Concepts

The principles of chemistry and biochemistry are applied in most areas of the biological sciences, including cell biology, metabolism and physiology, microbiology, genetics, and the regulation of each of these processes.

Cell Metabolism. Biochemical principles apply to metabolic pathways carried out in both eukaryotic cells (nucleated cells such as those of plants and animals) and in prokaryotic cells, such as bacteria or other microbes. Carbohydrates, proteins, and lipids are metabolized in specific pathways to generate compounds necessary for reproduction and growth as well as being sources of energy. Common to nearly all cells are the primary pathways such as glycolysis (the breakdown of simple sugars such as glucose to pyruvate) and the Krebs cycle (the further oxidation of pyruvate), generating both intermediates for other pathways and sources of energy through movement of electrons. Organisms that carry out respiration with oxygen as the terminal electron acceptor (plants, animals, and many types of bacteria) produce much of their energy “currency” in the form of ATP through a process called oxidative phosphorylation, which is the production of ATP through a shift of energy as molecules become progressively oxidized. Biochemistry involves the study of not only intermediates in these pathways but also enzymes that catalyze each step and of the regulation of those pathways.

Metabolic Regulation. Regulation of metabolic activity occurs largely through two processes: gene expression and production of enzymes. Gene expression in prokaryotes such as bacteria is commonly a “negative” process in which the regulator is a repressor that inhibits gene activity. The presence of an activator that binds the repressor, removing it from its site on the DNA, allows reexpression of the gene. An example is the regulation of the lactose operon in bacteria, a series of genes that regulate the uptake and breakdown of the sugar lactose. In the absence of lactose, a repressor blocks operon expression. When the cell is exposed to lactose, the sugar binds the repressor, altering its structure, which releases it from the DNA. The result is expression of the gene for an enzyme, beta-galactosidase, which hydrolyzes the lactose into the simpler sugars glucose and galactose, each of which enters glycolysis.

Regulation in eukaryotic cells involves “positive” control in which the regulator activates gene expression, usually through a multistep pathway beginning at the cell surface. An example of the process is regulation of cell replication. The process begins when a growth factor binds a receptor on the cell membrane. A cascade of reactions results that alters the structure of a specific DNA-binding protein: the activator for gene expression.

Metabolic regulation may also take place using the enzyme itself. The product of the reaction may provide feedback by binding a site on the enzyme that reduces its activity. Respiration pathways are regulated in this manner. The feedback mechanism may be competitive, in which the product resembles the initial substrate and competes for the active site on the enzyme (a process common in amino acid synthesis), or “allosteric,” in which the product binds an alternative site on the enzyme, changing its shape and reducing the activity. Many antibiotics function as competitive inhibitors of bacterial enzymes. Enzyme study generally involves the isolation and purification of the enzyme in question, which allows direct analysis of the molecule, the effect of various substrates, and the physical changes that take place as the enzyme interacts with other molecules.

Genetics. The source of hereditary information in both eukaryotic and prokaryotic cells is DNA. Some viruses use RNA for genetic information, but the principles of expression remain the same. Biochemical principles govern the expression of DNA in a manner likened to information flow: The genetic information in DNA is transcribed to RNA and then translated into proteins; this information flow is known as the “central dogma” of molecular biology. All processes in the cell are regulated by proteins. Mutations (alterations in the genetic information) may cause significant changes in the regulation of cell processes. Cancer, for example, is, at its core, a disruption of the biochemistry of cell pathways. An understanding of these pathways and their regulation is among the critical discoveries in cancer research taking place since the 1970s.

Biochemical Techniques. The evolution of molecular techniques was among the greatest changes that took place in the field of biochemistry during the twentieth century. The studies carried out during the first decades of the century involved the partial purification of the molecule from the cell, followed by treatment to determine its structure; in this manner, enzymes were determined to be proteins. The changes in chemical properties of the substrate allowed biochemists to observe events that took place during the reaction. In 1942, Edward Tatum and George Beadle addressed what a “gene” is, using mutants of the bread mold Neurospora that were unable to utilize pathways in the synthesis of amino acids. Their conclusion was that a gene encodes an enzyme, the “one gene–one enzyme hypothesis.” Their hypothesis was modified as it became clear that gene products are not only enzymes and that genes may also encode multiple proteins.

Biochemical methods developed since the 1970s have significantly “simplified” analysis of enzymes and their substrates. Whereas in earlier decades identification of compounds required comparison with known structures or an understanding of the chemical properties of molecules, techniques such as X-ray crystallography and methods of separation using chromatography have resulted in increased ability to visualize events at the molecular level.

Applications Past and Present

Structure and Function of DNA. A common misconception is that DNA, the genetic material for most forms of life, was discovered by James D. Watson and Francis Crick during the early 1950s. However, DNA was first isolated in 1869 by Swiss chemist Johann Friedrich Miescher, who named it “nuclein”; it was termed a nucleic acid because of its chemical properties. Until the 1920s, the chemical makeup of DNA was known only to the extent of its components—a five-carbon sugar, nitrogen-containing bases and phosphates—but its function was unknown. During the 1930s and 1940s, a biochemical team at the Rockefeller Institute (later Rockefeller University) led by Oswald Avery demonstrated that when DNA is hydrolyzed (broken down), solutions containing DNA lose their ability to change the genetic characteristics of cells; the team concluded correctly that DNA was the source of genetic information. In 1953, Watson, Crick, Rosalind Franklin, and Maurice Wilkins, relying largely on the technique of X-ray crystallography, determined the structure of DNA.

Cancer Chemotherapy. Increased understanding of metabolic pathways in cells coupled with serendipitous discoveries associated with the poisonous gasses of World War I led to chemotherapy as a treatment for cancer. Nitrogen mustards were initially developed as weapons, first by the Germans and later by the Allies. A German bombing of a ship docked in Italy during World War I released a cargo of mustard gas that had been stored on the ship. Physicians observed that in addition to casualties directly related to exposure to the mustard gas, persons exposed to the gas had bone marrow cell numbers that were significantly depleted. This knowledge was applied in treating victims of leukemia and lymphomas to reduce the level of neoplastic cells, representing the first use of chemotherapy in controlling cancer.

The discovery of some metabolic analogs, drugs that resembled intermediates in biochemical pathways but that would inhibit enzymes in those pathways, resulted in the development of folic acid analogs—aminopterin and methotrexate—for treatment of other forms of cancer. By the beginning of the twenty-first century, several drugs had been developed for treatment of various types of cancers, most based on the same principle of inhibition of enzymes or pathways necessary for tumors to develop.

Biochemical Basis of Cancer. The ability of filterable agents, subsequently shown to be RNA viruses, to produce tumors or leukemia when injected into animals had been known since the 1890s. The molecular basis remained unknown until the end of the twentieth century. During the 1960s, while studying biochemical events that took place after infection by these viruses, Howard Temin suggested replication takes place through a DNA intermediate that integrates into the cell chromosome; the enzyme known as a reverse transcriptase was discovered several years later. The genetic information in the virus that caused the cell transformation from normal to neoplastic became known as an oncogene. In the 1970s, it was discovered that oncogenes do not originate with the virus but are cell genes. Within twenty years, more than one hundred oncogenes were observed in cells, all related to regulation of cell replication.

Oncogene products were characterized according to their functions and were found to fall into four major categories: growth factors, growth factor receptors, enzymes involved in the signaling pathway, and DNA binding proteins. A separate category of oncogenes became known as tumor suppressor genes, molecules that function as “stop signs” by inhibiting cell replication. Transformation of normal cells into cancer requires the mutation or alteration of combinations of certain oncogenes, in effect causing a cell to “short circuit” in a way in which the cell loses control of the replication process. In some cases identical forms of cancers in different individuals result from specific types of mutations. For example, many forms of colon cancer originate with a mutation in the same signaling pathway within the cell. Many forms of prostate cancer are found to share a common deletion mutation, the loss of a portion of a gene, in the cells of the tumor. Eventually, it may be possible to monitor the risk for certain forms of cancer through genetic analysis of the genes in question. This type of monitoring is already possible for certain inherited forms of breast cancer in which mutations in one of two breast cancer genes, BRCA1 and BRCA2, place the woman at significant risk for early development of the disease.

In theory, the same form of genetic analysis could be applied in determining the risk for other forms of chronic illnesses, such as heart disease, dementias, or metabolic diseases, in which samples of a person’s genome could undergo analysis for mutations in the relevant sites. Proper regulation in applying any results would be necessary to avoid abuse by health-care or insurance companies.

Immunosorbent Assays for Measurement of Molecules. Prior to the 1960s, biochemical assays to determine the concentration of molecules were largely colorimetric—reagents reacted with each other to produce color changes that could be measured with spectrophotometers or other such instruments. Measurements could be carried out only if the concentration of the substance was relatively high, so such assays were not particularly useful for measuring substances such as proteins in the blood. During the early 1960s, Rosalyn Yalow and Solomon Berson developed a much more sensitive method of measurement in which they monitored the binding of radiochemicals to the substrate in question. A modified form of the radioimmunoassay, as it was called, utilized a competitive assay in which the substrate of unknown concentration would compete with a radioactive molecule for binding. Comparison with known standards would allow an accurate determination of minute quantities of the unknown substance. During the 1980s, the technique was further modified by replacing the radioactive molecule with an enzyme; the level of activity was a function of the concentration of the unknown substance. Eventually known as the enzyme-linked immunosorbent assay (ELISA), the technique is utilized by most hospital or biochemical laboratories for measurement of minute quantities of materials.

Social Context and Future Prospects

Biochemistry has evolved from an observational science to one that focuses increasingly on events at the molecular level. Improved technology and instrumentation has impacted both pure research and the application of the research, particularly in medicinal biochemistry. The first antibiotics, including penicillin and streptomycin, appeared on the popular market in the years after World War II; dozens followed, most the result of widespread screening of soil bacteria. Most of these substances function in inhibiting bacteria growth, and microbes have become increasingly resistant. New methods are necessary to develop the next generation of antimicrobials, and much of the research has involved “designer drugs,” chemicals artificially synthesized that focus on specific sites or chemical structures of bacteria.

With an aging population, chronic illnesses such as cancer, heart disease, and dementia have taken a toll on older people. Biochemical research has focused on better understanding aging effects and molecular changes at the cellular level as well as studies that attempt to slow or reverse such processes. Some dementias are confirmed only upon autopsy; improved methods of molecular detection of byproducts of nerve damage at earlier stages may provide a means to recognize or slow such processes.

Improved understanding of the cellular processes that occur during the transformation of normal cells into cancers has led to the realization that certain cancers are characterized by specific mutations at the cell surface or in regulation pathways. It was expected that biochemical development of anticancer drugs would increasingly target specific sites on or within cancer cells in hopes of treating the disease specifically, while minimizing any harmful side effects to surrounding tissues. Sensitive assays remained necessary to discover or monitor molecules or pathway intermediates specific to the illness.

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