What are enzymes? |

Structure and Functions

Enzymes are remarkable molecules because they increase rates of biochemical reactions. Each enzyme within a cell selectively speeds up, or catalyzes, one particular reaction or type of reaction. The vast majority of enzymes belong to the class of large molecules known as proteins. Proteins are built by combining amino acids. There are twenty amino acids, which can be divided into three classes: hydrophobic, charged, and polar. Hydrophobic amino acids behave chemically like oils, avoiding contact with water. Charged amino acids are ionic, containing one extra or one less electron than do neutral molecules. Polar amino acids are attracted to water and other polar amino acids. Each of these three classes of amino acids has a distinct chemistry. The specific order of the amino acid sequence defines the structure and function of every protein. Inside cells, enzymes catalyze reactions so that they occur millions of times faster than they would without the presence of these proteins. Each cell in the body produces many different enzymes. Different sets of enzymes are found in different tissues, reflecting the specialized function of
each particular enzyme. Thousands of different enzymes are at work in the body; many have yet to be discovered.

Protein enzymes work by bringing the reactants in a chemical reaction together in the most favorable geometrical arrangement, so that bonds can be easily broken and reformed. This is possible because different enzymes have different three-dimensional shapes. It is the shape of the enzyme that determines its chemistry. Each enzyme combines with a specific substrate, or reactant, and catalyzes its characteristic reaction. When the reaction is over, the substrate has been converted into products. The enzyme remains unchanged, ready to catalyze another reaction with the next substrate molecule it encounters.

Enzymes play a significant role in treating diseases. Because enzymes have specific functions, a particular enzyme that has the required function to treat the disorder can be administered. Modern methods of genetic engineering allow the production of desired enzymes. Scientists can use bacteria as factories to produce large amounts of enzyme from an organism by copying the gene from the organism of interest into bacterial cells. The bacteria are then grown in culture, producing the enzyme of interest as they grow. This procedure is a much safer method than the old procedure of isolating enzymes from animal tissues, because the enzymes produced are free of viruses and other contaminants present in animal tissues. Proteins produced by genetic engineering techniques are called recombinant proteins.

Sometimes enzymes can be used as drugs for the treatment of specific diseases. Streptokinase is an enzyme mixture that is useful in clearing blood clots that occur in the heart and the lower extremities. Another useful enzyme for dissolving blood clots that occur as a result of heart attacks is human tissue plasminogen activator (TPA). Recombinant TPA is produced by genetic engineering techniques, using bacteria cultures to produce large quantities of human TPA. The administration of TPA within an hour of the formation of a blood clot in a coronary artery dramatically increases survival rates of heart attack victims. Some types of adult leukemia are treated by intravenous administration of the asparaginase enzyme. Tumor cells require the molecule asparagine to grow, and they scavenge it from the bloodstream. Asparaginase drastically reduces the amount of asparagine in the blood, thus slowing the growth of the tumor. Because most enzymes do not last long in blood, huge amounts of enzymes are required for therapeutic effects. In classic
hemophilia, the factor VIII enzyme is missing or is genetically mutated so that it has a very low activity. This enzyme is essential for inducing the formation of blood clots. In the past, it was a laborious task to collect a concentrated blood plasma sample containing factor VIII, which was administered to hemophiliacs to stop hemorrhages. This treatment carried the risk of infecting the patient with viruses that cause acquired immunodeficiency syndrome (AIDS), hepatitis, and other diseases. Purified recombinant factor VIII is now available. Because the recombinant human factor VIII is produced by bacteria, it cannot be infected with the viruses that cause hepatitis and AIDS.

A classic enzyme inhibitor used as a drug is penicillin. Penicillin was discovered in 1928 by Alexander Fleming, after he noticed that bacterial growth was prevented by a contaminating mold known as Penicillium. Ten years later, Howard Florey and Ernst Chain performed the key experiments that led to the isolation, characterization, and clinical use of this wonder drug antibiotic. In 1957, Joshua Lederberg showed that penicillin interferes with the synthesis of the cell walls of bacteria. In 1965, James Park and Jack Strominger independently discovered that penicillin blocks the last step in cell wall synthesis. The last step is the cross-linking of different strands of the wall and is catalyzed by the enzyme glycopeptide transpeptidase. The shape of penicillin resembles that of the normal substrate of glycopeptide transpeptidase, so that penicillin binds to the active site of the transpeptidase enzyme. Once bound to the active site, penicillin forms a permanent bond with one of the amino acid residues. This chemical reaction permanently inhibits the glycopeptide transpeptidase enzyme, thus preventing the transpeptidase from cross-linking the bacterial wall.

Several anticancer drugs work by blocking the synthesis of deoxythymidylate (dTMP), as an abundant supply of dTMP is required for rapid cell division to be sustained. Drugs that inhibit the enzymes thymidylate synthase and dihydrofolate reductase are very effective agents in cancer chemotherapy. Thymidylate synthase, which makes dTMP from deoxyuridylate, is irreversibly inhibited by the drug fluorouracil. This drug is converted into fluorodeoxyuridylate (F-dUMP), which chemically reacts with thymidylate synthase so that the enzyme can no longer function in its normal role of making dTMP from deoxyuridylate. The synthesis of dTMP can also be blocked by drugs that inhibit the enzyme dihydrofolate reductase. The normal substrate for dihydrofolate reductase is the molecule dihydrofolate. Drugs such as aminopterin and methotrexate bind to the active site of the reductase enzyme, inhibiting rapid cell growth. Methotrexate is very effective at inhibiting rapidly growing tumors such as acute leukemia and choriocarcinoma. Unfortunately methotrexate kills all rapidly dividing cells, including stem cells in bone marrow, epithelial cells of the intestinal tract, and hair follicles, which explains the many toxic side effects of this drug. Computer-aided drug design has been applied to the dihydrofolate reductase enzyme, with encouraging results.

The activity of an enzyme is a measure of how efficiently a particular enzyme catalyzes its reaction. A loss in activity corresponds to a decrease in catalytic efficiency, and an increase in activity corresponds to an increase in catalytic efficiency. Many drugs increase enzyme activity (enzyme induction), and many decrease enzyme activity (enzyme inhibition). Both enzyme induction and enzyme inhibition result from the interaction of the drug with the enzyme, altering the surface of the enzyme where the substrate normally is bound during catalysis. In enzyme induction, the surface is altered such that the substrate is bound tighter than usual, while in enzyme inhibition, the surface is altered so that the substrate cannot bind to the enzyme. The structures of many enzyme inhibitors are similar to the structures of substrates. Inhibitors bind at active sites of enzymes. Drugs that are enzyme inhibitors are very powerful medical tools, as they bind to the enzyme and are not easily removed.

Universities, government agencies, and pharmaceutical companies are continually seeking to develop drugs that specifically bind and inhibit enzymes responsible for disease. Much effort is spent trying to design drugs in a rational manner, using the most powerful tools of chemistry. Techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and computational chemistry allow researchers to determine the shapes of enzymes, their substrates, and their inhibitors. These efforts allow the research team to design drugs that bind more specifically to the target enzyme, thus increasing the effectiveness and lowering the toxicity of the drug.

Disorders and Diseases

Defects in enzymes, known as mutations, can cause disease. A protein molecule is mutated when one or more of the original amino acids in the protein is replaced by a different amino acid. For example, if an enzyme consists of one hundred amino acids, and amino acid number 35 is changed from one kind of amino acid to a different kind, the protein is now a mutant. A mutated enzyme has a slightly altered shape compared to the original enzyme. If the change in shape causes the enzyme to perform its chemistry more slowly than the original enzyme, then the cell and tissue have an impaired function. In particular, if an amino acid is changed from one of the three classes (hydrophobic, charged, or polar) to a different class, then the mutation is more likely to cause a change in the structure and function of the enzyme. Not all mutations are harmful, but a single mutation in a key region of an enzyme can be fatal to a living organism.

Many diseases are diagnosed by measuring enzyme concentrations and activities in the body. Enzyme concentration refers to the amount of enzyme present, while enzyme activity refers to the ability of the enzyme to perform its chemistry. Enzyme concentrations and activities can be measured in blood or in tissue. Disease of tissues and organs can cause cellular damage, so that enzymes that are normally not present in significant quantities in blood are raised to very high levels as they flow from the damaged tissue into the blood plasma. Detection of particular enzymes in blood plasma indicates a diseased organ. The higher the concentration of enzyme in the blood, the more extensive the damage to that tissue or organ. The detection of these enzymes in the blood is a diagnostic tool, indicating a particular disorder. Genetic diseases caused by a mutation in an enzyme can be detected by laboratory tests that measure enzyme activity or enzyme shape.

Disease diagnosis is often made by measuring the concentration or activity of enzymes. Isozymes are enzymes that catalyze the same reaction but have slightly different structures. Most isozymes are enzymes consisting of two or more subunits, with different combinations of the subunits differentiating the isozymes. Isozymes of the enzymes lactate dehydrogenase, creatine kinase, and alkaline phosphatase are used for clinical applications. Monitoring of the isozyme concentrations and activities of lactate dehydrogenase and creatine kinase in the blood shows whether a patient has suffered a heart attack.

Creatine kinase consists of two subunits. The two possible subunits are M, which stands for muscle type, and B, which stands for brain type. There are three possible isozymes: MM, BB, and MB. The MM isozyme consists of two M subunits and is the only isozyme found in skeletal muscle, the BB isozyme consists of two B subunits and is the only isozyme found in the brain, and the MB isozyme consists of one M and one B subunit and is found only in the heart. Lactate dehydrogenase consists of four subunits, made from five combinations of two subunits. The two subunits are the heart subunit, designated by H, and the muscle subunit, designated by M. The HHHH and HHHM isozymes are found in the heart and in red blood cells, the HHMM isozyme is found in the brain and kidney, and the MMMM isozyme is found in the liver and skeletal muscle.

After a heart attack, the cellular breakup of heart tissue releases the MB isozyme of creatine kinase into the bloodstream within six to eighteen hours. Release of lactate dehydrogenase into the blood is slower than that of creatine kinase, occurring one to two days after the appearance of creatine kinase. In a healthy person, the activity of the HHHM isozyme of lactate dehydrogenase is higher than that of the HHHH isozyme. In heart attack victims, however, the activity of the HHHH isozyme becomes greater than that of the HHHM isozyme between twelve and twenty-four hours after the attack. Measurement of increased concentration of the MB isozyme a short while after a suspected heart attack, followed by the switch in lactate dehydrogenase activity between the HHHH and HHHM isozymes, indicates that a heart attack occurred. Secondary complications of a heart attack can also be followed with isozyme measurements. For example, increased activity of the MMMM isozyme of lactate dehydrogenase is an indication of liver congestion.

Certain medical conditions can be screened by using immobilized enzymes as reagents in desktop clinical analyzers. For example, screening tests for cholesterol and triglycerides can be completed in a few minutes using 0.01 milliliter of blood plasma. The enzymes cholesterol oxidase and lipase are immobilized, or fixed in place, in a detection kit. If cholesterol is present, cholesterol oxidase breaks off hydrogen peroxide from the cholesterol. The enzyme peroxidase and a colorless dye are included in the detection kit, and peroxidase catalyzes the reaction of the colorless dye and hydrogen peroxide to form a colored dye that can be easily measured from the amount of light reflected from the solution. The enzyme lipase allows the accurate determination of triglycerides in blood.

A mutation in a protein that acts as a natural inhibitor of an enzyme can cause disease. For example, emphysema is a destructive lung disease in which the alveolar walls of the lungs are destroyed by an enzyme known as elastase. A person with emphysema breathes much harder to exchange the same volume of air because the alveoli, or air pockets, have become much less efficient. Normally, the elastase enzyme is prevented from destroying lung tissue by the protein antitrypsin. Antitrypsin is made in the liver and flows to the lungs, where it binds to the active site of elastase and prevents it from digesting lung tissue. Emphysema can occur when the negatively charged amino acid at position 53 of the amino acid sequence of antitrypsin is replaced with a positively charged amino acid. This mutation changes the chemical nature of antitrypsin such that the mutant antitrypsin is released from the liver at a much slower rate. The level of this mutant antitrypsin in the lungs is 15 percent of the normal level. The net result of this one amino acid mutation in the antitrypsin protein
is that most of the elastase enzyme is free to destroy lung tissue. Cigarette smoking dramatically increases the incidence of emphysema in people who have the mutant antitrypsin. Cigarette smoke reacts with the hydrophobic amino acid at position 358 of antitrypsin, adding one oxygen atom at this position in the amino acid sequence. The addition of this one extra oxygen atom at this critical place in antitrypsin changes the chemical nature of the hydrophobic amino acid so that the antitrypsin no longer can bind to elastase. Because only 15 percent of the mutant antitrypsin gets from the liver to the lungs in the first place, cigarette smoking puts people with this particular mutation at grave risk for developing emphysema.

Perspective and Prospects

Enzymatic reactions have been used by humankind since prehistoric times. It has been known for more than six thousand years that fermentation processes transform grapes into wine, but it was not until the nineteenth century that it was understood that the conversion of grape sugar to alcohol is a process catalyzed by enzymes found in yeast. In the eighteenth century, Antoine Lavoisier showed that a solution of sugar could be fermented if provided with the sediment of a previous fermentation and that the sugar was converted to alcohol and carbon dioxide in this process. At this time, it was thought that there was a vital force responsible for the workings of a living cell. This notion of a vital force slowed the development of the discipline of biochemistry considerably, as many good scientists struggled to understand the fermentation process. In 1828, Friedrich Wöhler synthesized urea in a test tube, providing strong evidence against the concept of a vital force. In 1833, Anselme Payen and Jean Persoz discovered
the first enzyme, diastase (now known as amylase), which converted starch into sugar. The next year, Johann Eberle showed that the presence of a stomach is not required for gastric
digestion to take place. In 1836, Theodor Schwann made the very important discovery that the active ingredient in digestion, which he called pepsin, could be extracted from the stomach wall.

The next year, Jöns Jakob Berzelius developed the idea of catalysis, making the point that both living and inorganic systems had catalysts. In the late 1850’s, Louis Pasteur confirmed and extended the earlier experiments of Schwann. Despite his brilliant experimental abilities, however, Pasteur was handicapped in his research by his belief that fermentation could happen only within a living organism. In 1860, Marcelin Berthelot showed that a living being was not the ferment, but produced the ferment, in sharp contrast to Pasteur’s vitalist ideas. Pasteur’s response to this work was that Berthelot and he meant different things by the use of the word “ferment.” Moritz Traube, a German wine merchant, realized that chemical processes and living bodies were mostly based on ferment actions, and he published these ideas in 1858 and again in 1878. In 1878, Friedrich Kühne proposed that to remove the discrepancy over the meaning of the word “ferment,” the word “enzyme” should be used, as it means “in yeast.” It was not until 1897 that Eduard Buchner showed that living cells are not essential for fermentation to occur, as he extracted from yeast a cell-free juice containing the entire fermentation system.

From 1894 to 1898, Emil Fischer used synthetic organic chemistry for the preparation of substrates of known structure and configuration. He showed that enzymes have a very high degree of specificity for their own particular substrate and developed the famous “lock-and-key” hypothesis. This theory, which has been only slightly modified, states that the shape of a substrate and the enzyme’s active site must be complementary for catalysis to occur. Purification of enzymes remained a difficult problem, and it was not until 1926 that James Summer crystallized the first enzyme, jack bean urease. The sequence of protein enzymes could be determined experimentally after 1952, when Frederick Sanger developed his methods for amino acid sequencing. In 1965, David Phillips produced the first three-dimensional picture of an enzyme, determining the shape of lysozyme. The advent of genetic engineering techniques in the 1970’s revolutionized the field of enzyme research and the use of enzymes in medical applications by enabling the production of
copious amounts of recombinant proteins.

Bibliography

Campbell, Neil A., et al. Biology: Concepts and Connections. 6th ed. San Francisco: Pearson/Benjamin Cummings, 2008. This classic introductory textbook provides an excellent discussion of essential biological structures and mechanisms. Its extensive and detailed illustrations help to make even difficult concepts accessible to the nonspecialist. Of particular interest is the chapter on enzymes, titled “An Introduction to Metabolism.”

Copeland, Robert A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2d ed. New York: Wiley, 2000. An introductory text that examines the structural complexities of proteins and enzymes and the mechanisms by which enzymes perform their catalytic functions.

Fruton, Joseph S. Molecules and Life. New York: Wiley-Interscience, 1972. Fruton, a Yale biochemist, has filled his book with historical essays on the interplay of chemistry and biology. The first part of the book, “From Ferments to Enzymes,” is an interesting account of how science progressed from the known results of fermentation to the chemical knowledge that enzymes were the molecules responsible for this and all other biochemical processes.

Kornberg, Arthur. For the Love of Enzymes: The Odyssey of a Biochemist. Cambridge, Mass.: Harvard University Press, 1989. Both an autobiography of a great biochemist and a history of the study of enzymes. Arthur Kornberg won a Nobel Prize for the laboratory synthesis of deoxyribonucleic acid (DNA). An excellent scientific biography.

Liska, Ken. Drugs and the Human Body, with Implications for Society. 8th ed. Upper Saddle River, N.J.: Pearson/Prentice Hall, 2009. An easy-to-read book about the effects of drugs on the human body. A good overview of how drugs interact with various molecules in the body, including many cases in which enzymes are drug targets.

Palmer, Trevor. Understanding Enzymes. 4th ed. New York: Prentice Hall, 1995. A standard text on enzymes and how they function. Includes a bibliography and an index.

Silverman, Richard B. Organic Chemistry of Enzyme-Catalyzed Reactions. Rev. ed. San Diego, Calif.: Academic Press, 2002. A text that examines the general mechanisms used by enzymes and stresses that enzymology is simply a biological application of physical organic chemistry.

Voet, Donald, and Judith G. Voet. Biochemistry. 3d ed. Hoboken, N.J.: John Wiley & Sons, 2004. A text that approaches biochemistry via organic chemistry reactions.