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Chapter 1: Actions and Reactions
Even though you're probably sitting down while you're reading this, your body is anything but static. Thousands of enzymes in your body toil away every second of every day, breaking apart the components of the foods you eat into energy for essential life processes. Vision, movement, memory—you name it, there are enzymes at work behind the scenes.
Enzymes work by making it possible for chemical reactions inside your body to take place. While that might not seem significant, consider the fact that without the help of enzymes, the conversion of nutrients and minerals into usable biological molecules such as proteins and nucleic acids might take weeks, even years. Enzymes can make this happen in minutes, sometimes seconds.
How do they do it all, and so well? Enzymes act like the accelerator pedal of a car. But they also play the role of matchmaker, bringing together starting materials (called substrates) and converting them into finished materials (called reaction products). One secret to an enzyme's success in this endeavor is its shape. An enzyme is shaped so that it can hug its substrate tightly. This molecular embrace triggers chemical changes, shuffling chemical attractive forces called bonds and producing new molecules. Only enzymes that have an exact fit with their substrates do a decent job of speeding up chemical reactions. But things don't end there; reactions are not singular events. They re-occur, over and over again. Enzymes are the key players linking up chain reactions of the chemical events that culminate in our everyday physiology. Much like a cascade of dominoes, the product of one chemical reaction becomes the substrate for another. Enzymes form the core of these ordered pathways, which themselves are the basis for metabolism. In a grand sense, metabolism is the process any organism uses to retrieve energy from Product the environment and use it to grow. The proper functioning of small and big body parts hinges upon effective communication within and between pathways. That includes everything from tiny specks of DNA that string together into all of your genes to a complicated, multicelled organ such as the heart. By understanding the language of physiological communication systems, scientists can devise ways to patch the circuits when they become broken, in illness and disease.
What Is Biochemistry?
bi•o•chem•is•try (bi ´ o kem ´ is tree):
n., the chemistry of living organisms.
Simply stated, biochemistry is life. Practically stated, biochemistry is our life: what we are and how we live. Our bodies are very busy factories, extracting energy from the foods we eat, building cells and tissues, and knitting everything together into a functioning unit using molecular tools called enzymes. Creatures as distinct as bacteria, giraffes, and people use many of the same biochemical toolsets to survive, eat, move, and interact with their respective environments. Biochemistry underlies our health.
The Motion of Life
Fish swim, birds fly, babies crawl. Enzymes, too, are constantly on the move. The world's smallest motor, in fact, is an enzyme found in the powerhouse of the cell (the mitochondrion) which generates energy in the form of a molecule called adenosine triphosphate, or ATP. Often dubbed the energy currency of life, ATP shuttles to and fro throughout cells, and is "traded" during chemical reactions. These molecular transactions drive reactions forward to make a product. Several decades of work earned three scientists—Paul Boyer of the University of California, Los Angeles, John Walker of the Medical Research Council in the United Kingdom, and Jens Skou of the University of Aarhus in Denmark—the Nobel Prize for figuring out how the motor, a molecule called ATP synthase, functions as a set of molecular levers, gears, and ratchets. Other molecular motors include protein machines that tote DNA-laden chromosomes or protein cargo throughout the cell. The enzyme that copies DNA does its job through what scientists call a "sliding clamp" mechanism. As a growing DNA strand, a future gene winds through an enzyme called DNA polymerase like a thread through a needle. The energy source for this and all biological motor-driven processes is ATP.
It's a Gas!
Tediously long car trips elicit multiple rounds of word games like "20 Questions," in which players take turns thinking of a secret object and having their opponent ask more and more detailed questions to identify it. In this game, the first question is always, "Animal, Vegetable, or Mineral?" Virtually everything imaginable falls into one of those three categories.
Chemists could play such a game, in which the mystery item is a molecule—any molecule. In this game, the first question would always be, "Solid, Liquid, or Gas?" Looking outside the window, it's easy to think of entries for each of these categories: Stones are solids, dewdrops are liquids, and the atmosphere is a blend of many different gases. But there's a trick: In theory, any molecule could in fact be all three, if the conditions were right. In pure form, whether a molecule is solid, liquid, or gas depends on its environment, namely the ambient temperature and the atmospheric pressure. Water is an easy example. Everybody knows that below 32 degrees Fahrenheit, water is a solid, and above 32 degrees, water is a liquid. Put a tea kettle on the stove and witness water turn into a gas.
Our bodies, too, harbor an assortment of solids, liquids, and gases. But of these three physical states, which chemists call phases, everything that lives is largely liquid—water is the universal solvent of life. Fingernails, hair, and bones are solids, indeed, but only the dead parts. Living cells resident in bone and its vital bone marrow thrive in a watery environment. Gases can also be found throughout the body; some examples include the oxygen we breathe in and the carbon dioxide we breathe out. But inside the body, even these gases are dissolved in liquids—mainly blood, which itself is mostly water.
One gas, nitric oxide (whose chemical symbol is "NO"), serves the body in a host of useful ways. Scientists were rather surprised not many years ago when they discovered that the gas NO is a chemical messenger. Tiny and hard to study in the laboratory, NO eluded scientists for many years. Other molecular messengers—such as neurotransmitters and larger proteins—can be relatively easily extracted from body fluids and studied in a test tube, where they can remain intact for minutes or even hours at body temperatures. NO, on the other hand, vanishes in seconds. While this property makes it horrendously difficult to study, such volatility renders NO a molecule with extraordinary versatility. In a snap, NO can open blood vessels, help pass electrical signals between nerves, or fight infections.
But in addition to being a friend, NO can also be a foe—too much or too little of this gas can be harmful. Blood vessels that have been widened too much can lead to potentially deadly shock, a condition in which blood pressure plunges so low that vital organs cannot get enough blood to survive. An overactive immune response—fired up by NO—can produce a painful syndrome called inflammatory bowel disease. With all this at stake, the body works hard to stringently control production of this powerful gas.
Stuck in a Corner?
The stereotypical image of a scientist as a faceless white coat hunched over a microscope in the corner of a lab has never been accurate, but it's even more wrong today. While plenty of scientists spend a lot of time in their own workspaces, they rely heavily on interaction with other scientists to share ideas and validate concepts—at scientific conferences, at the coffee maker, or via e-mail. Although a scientist in his or her own lab may crack away for years at a very small piece of a big puzzle—say, how a particular enzyme works—he or she spends much of the time communicating with other scientists to further the understanding of molecular secrets.
Chemical Biology in Action: Just Say NO
T.L. POULOS, H. LI, C.S. RAMAN
The molecule that manufactures NO is an enzyme called nitric oxide synthase (NOS). Owing to nitric oxide's many different functions in the body, three different versions of NOS exist, specialized for the cardiovascular, immune, and nervous systems. In recent years, scientists have achieved a major victory in beginning to understand how NOS works. Thomas Poulos of the University of California, Irvine, determined the structure, or three-dimensional shape, of one form of NOS. Since intimate associations between an enzyme and its substrate rely on a snug fit, probing the three-dimensional shape of an enzyme or other protein can enable scientists to begin to understand how the protein works, predict what other molecules it might fit, and design drugs to boost or block its activity. After obtaining a sample of NOS protein from the laboratory of Bettie Sue Masters of the University of Texas Health Science Center at San Antonio, Poulos obtained a "picture" of NOS by bombarding a tiny crystal arrangement of the protein with high-energy X rays, then piecing together the protein's shape by tracing the directions in which the energy was scattered throughout the crystal. This work, years in the making, paints a portrait of NOS consisting of two identical units. In a cell, the two units of NOS assemble head-to-head, creating a new landscape upon which substrates and helper molecules convene to complete the task at hand: creating nitric oxide from an amino acid called arginine. In the case of NOS, the helpers include iron and a tiny molecule called a cofactor. Enzymes like NOS are lost without these helpers.
Folic Acid Saves the Day
In
baking, some ingredients are simply not optional—forget the baking
powder, forget the muffins. Just as baking powder is essential for some
recipes, helper molecules called cofactors are necessary ingredients for
many biochemical reactions.
Take folic acid (one of the B vitamins), for example. Scientists have known for decades that folic acid can protect against certain birth defects—such as spina bifida—that develop during the first few weeks after conception. For this reason, the Food and Drug Administration recommends that every woman of child-bearing age supplement her diet with 400 micrograms of the vitamin. Scientists figured out that folic acid does its molecular good deeds by lowering levels of a potentially harmful compound called homocysteine, which is also a risk factor for heart attacks and strokes. As it turns out, folic acid performs this task by speeding up the conversion of homocysteine to methionine, a nontoxic amino acid that the body needs.
Folic acid does this by improving the fit between an enzyme and its cofactor. The enzyme in this case is known shorthand as MTHFR, and the cofactor, a molecule called FAD, is also vitamin-derived (from vitamin B2) and is essential for converting homocysteine to methionine. Martha Ludwig and Rowena Matthews, both of the University of Michigan Medical School in Ann Arbor, determined that by locking FAD onto MTHFR, folic acid performs this protective role in the body.
Pulling Into Dock
Like a ship nestling into its berth, many proteins require the help of one or more other proteins to perform their jobs well. However, unlike ships, proteins docked together often change their shape as a result of such an encounter. The differently shaped protein is newly and exquisitely able to capture a substrate and carry out a chemical reaction. Akin to rearranging seats in a room to accommodate more guests, the reshaping of proteins (called conformational changes) can make extra room for substrates and products to fit. Such shape changes also change the electrical "ambience" of an enzyme's innards, revealing differently charged portions of the molecule that can have a big impact on molecular interactions.
Making a Protein From Scratch
Tucked away inside the DNA sequence of all of your genes are the instructions for how to construct a unique individual. Our genetic identity is "coded" in the sense that four building blocks, called nucleotides, string together to spell out a biochemical message—the manufacturing instructions for a protein. DNA's four nucleotides, abbreviated A, T, G, and C, can only match up in specific pairs: A links to T and G links to C. An intermediate in this process, called mRNA (messenger ribonucleic acid), is made from the DNA template and serves as a link to molecular machines called ribosomes. Inside every cell, ribosomes read mRNA sequences and hook together protein building blocks called amino acids in the order specified by the code: Groups of three nucleotides in mRNA code for each of 20 amino acids. Connector molecules called tRNA (transfer RNA) aid in this process. Ultimately, the string of amino acids folds upon itself, adopting the unique shape that is the signature of that particular protein.
Building Blocks
So the case is made that enzymes, and all proteins, are extremely important in the body. Where do these important molecules come from? Do they last forever?
Proteins are synthesized continually throughout life. Stockpiles of proteins are not passed on from generation to generation, but their molecular instruction guides—our genetic material, DNA—are. After reading the DNA "letters" in our genes, specialized molecular machines (groups of enzymes working side by side inside the cell) copy the DNA, then other machines use this genetic template to churn out proteins. To do this, enzymes mix and match a set of 20 different amino acids, the building blocks of proteins. Hooking together these amino acids, the body constructs thousands of different protein types. Theoretically, millions of proteins could be formed through all the possible linkages between amino acids. It is not surprising, then, that every one of these amino acids must be readily available at all times for protein synthesis.
Dire consequences may result if one or more of these amino acids is either absent or overabundant. For instance, a genetic disorder called phenylketonuria (PKU) is caused by the body's inability to get rid of extra phenylalanine, an amino acid abbreviated "Phe." PKU is an "autosomal recessive" disorder, meaning that the only way to get the disease is if both of your parents carry a version of a gene linked with this disease. If only one parent has the gene linked to PKU, his or her children cannot develop the disease. Children who have PKU are born without the enzyme that breaks down the Phe amino acid. Extremely high levels of Phe accumulate and are very toxic, especially to the brain. As a result, PKU causes mental retardation. Yet Phe is an essential amino acid—your body cannot do without it. Both diet and genes contribute to causing PKU, and so any means to control the supply of Phe in the body can prevent the disease.
A thin silver lining to the PKU story is that the disorder can be diagnosed simply—in fact, since the 1960s, nearly every baby born in the United States has received a tiny needle stick in his or her heel to retrieve a droplet of blood to test for levels of the Phe-chewing enzyme. If caught early enough and treated in the first year of life, PKU can be controlled. In 150 million infants tested since the early 1970s, 10,000 cases of PKU have been detected and treated. At present, doctors treat children with PKU by prescribing a life-long restrictive diet; certain foods, such as milk and diet sodas containing the artificial sweetener aspartame (NutraSweet®), are rich sources of Phe. The diet is rigid, requiring children to avoid those and many other foods, such as meat and fish, dairy products, bread, nuts, and even some vegetables. As a result, people with PKU have to take a special Phe-free vitamin/mineral supplement to ensure that they receive adequate amounts of all of the other essential amino acids bountiful in those foods. People used to think that once a child with PKU reached the teens, he or she could go off the diet, which can be expensive because of the supplement. However, current guidelines recommend that people with PKU remain on the restrictive diet throughout life.
To get around the difficulty and inconvenience of maintaining a highly specialized diet and taking a dietary supplement for life, a better solution might be to provide the Phe-digesting enzyme to people whose bodies lack it. But while replacing the missing enzyme that breaks down Phe (abbreviated "PAH") may seem a simple plan, this is much easier said than done. The PAH enzyme has many parts and cofactors. What's more, delivering the enzyme requires a liver transplant, a procedure that itself carries significant risks. An alternative approach would be to supply people with PKU with an enzyme that will get rid of Phe and that can be safely administered by mouth. Such an enzyme, called "PAL," abounds in nature—plants, yeast, and a variety of other organisms have it, and scientists can produce it in the lab using genetic engineering strategies. Recently, researchers have succeeded in treating an experimental strain of mice who develop a PKU-like syndrome with lab-made PAL. Clinical studies in people will determine for sure whether this strategy offers hope for people with the disease.
A Special Bond
You may be surprised to learn that at the heart of chemistry is physics—the study of attracting and repelling forces that link up the building blocks of life. Chemical bonds are those physical forces that keep atoms together, and they come in a few varieties (see drawing/illustration at right). Ionic bonds, in which positively charged atoms are attracted to negatively charged atoms, are the strongest of the bond types. Covalent bonds are more subtle, and occur when neighboring atoms (such as hydrogen) share electrons from within their respective halos of swirling particles. Chemists refer to both ionic and covalent bonds as "intramolecular" forces. Other important forces are called "intermolecular" forces—those holding different molecules together. These types of forces form the basis for liquids and solids, which are really just collections of molecules arranged in a precise pattern in space. Intermolecular forces are also called van der Waals forces, named for the Dutch physicist who first discovered them. Hydrogen bonds are a type of van der Waals force, and represent an important bond in biochemistry.
From Mice to...Bacteria?
A favorite experimental tool of many scientists is the laboratory mouse, which can be bred "to order" with characteristics useful for addressing specific research questions. While rodents differ from people in important and obvious respects, believe it or not mice and rats share many of the same genes with humans. In some cases, upwards of 80 percent of the nucleotides in a mouse gene may be identical to a similar one—its "homologue"—in humans. Nature is economical: Very important genes (those that code for key metabolic enzymes, for instance) are conserved throughout evolution, varying little between species. For researchers, that's a good thing. Mice and a slew of other so-called model organisms—such as bacteria, yeast, and even plants—are the workhorses of many biochemical laboratories. But in addition to these often striking similarities, there are significant differences in the biochemistry of model organisms, especially in the most primitive of species like bacteria. Scientists can exploit these differences to fight disease, targeting enzymes or other molecular parts that are common to microorganisms but are absent from your body.
A Killer's Strategy
A type of bacteria called enterococci possess enzymes that weave together alternating protein-sugar strands to create a tough cell wall—the bacteria's main defense against the outside world. The antibiotic drug vancomycin interferes with production of the cell wall (see drawing/illustration at left). This kills the bacteria, which stops infection. Some bacteria become resistant to vancomycin, and thrive in the presence of the antibiotic. The developing cell wall of vancomycin-resistant bacteria has a slightly different composition that the drug molecule does not recognize. Specialized resistance genes provide the resistant bacteria with the ability to reprogram the composition of the cell wall.
Chemical Biology in Action: Chemistry to the Rescue
Arguably many of the most important medical advances this century relate to the development of powerful antibiotics and vaccines to treat infectious diseases caused by bacteria, viruses, and parasites. But those breakthroughs have come with a cost—the microbes have learned how to fight back, and with a vengeance. The misuse of antibiotics—these drugs are overprescribed by doctors and people often fail to finish a full prescription—is the most common reason why antibiotic resistance is coming so rapidly to the fore.
When you take an antibiotic, the drug treats infection by knocking out hundreds of strains of "sensitive" bacteria in your body. But it also leaves behind scores of "resistant" strains—slightly altered versions of the sensitive variety. The resistant microbes, with no stops in place, repopulate themselves rapidly. To make matters worse, these lingering resistant organisms hang out not only in your body, but they can spread to your family and friends—worsening the problem for everyone.
Bacteria are not inherently malicious. In the human body, many different types of bacteria reside within the large intestine, where they perform vital roles in processing food. Trillions of microorganisms break down undigested carbohydrates, common components of vegetables and other foods like beans. In the wrong place, however, these normally innocuous bacteria—called enterococci—can do the body great harm. In disease, such microbes can seep from the relatively safe harbor of the intestines into other regions of the body, such as burned skin, the heart, or the urinary tract. There, the bacteria can multiply rampantly, especially when the immune system is already strained. Enterococci are stubbornly resistant to most antibiotic drugs. Until recently, an antibiotic called vancomycin fairly effectively put the brakes on enterococcal infections. However, in recent years the incidence of enterococcal resistance to vancomycin has been on a disturbing rise.
Fortunately, chemists are hot on the heels of enterococci. Christopher T.Walsh and Daniel Kahne, both of Harvard Medical School in Boston, Massachusetts, have traced the roots of vancomycin resistance to a single, errant chemical link. Vancomycin normally kills enterococci by getting in the bacterium's way while it tries to manufacture a protective cell wall for itself. Vancomycin prevents the molecular "bricks" of this cell wall from melding together, leaving the bacterium susceptible to the harsh environment and destructive enzymes in the cells of its host's body. Walsh, Kahne, and their coworkers unearthed a set of just five genes that enable enterococci to get past the antibiotic drug vancomycin's action by using a slightly different method to build a cell wall. The researchers' detective work points to promising avenues for future antibiotic drug development, based on the strategy of interrupting enzymes that rearrange the cell wall precursors.
Question Box: Computer Labs
In the future, computer models will be used to design experimental programs in research and industry...
Bioengineer Christophe Schilling works with computers, and he works in a lab. But his is not a garden variety computer lab. Schilling, who earned his Ph.D. in bioengineering from the University of California, San Diego, studies bacterial cells—and the thousands of enzymatic reactions occurring inside them—without ever putting on a pair of lab gloves or pouring bacterial broth into a flask. Schilling's in silico (literally, "within silicon," a component of computer hardware) research relies on computers to study how enzymatic pathways talk to each other and work together. But while the technique promises to be inordinately powerful in predicting cellular performance under a vast set of conditions, Schilling admits that he'd be out of a job if "wet" (laboratory-based) biochemists didn't keep busy in their labs figuring out what enzymes in the cell actually do. The Chemistry of Health asked Schilling about the potential of mapping a cell using computers.
CH: What don't we already know about what goes on inside living cells?
Schilling: Biologists are now forced to confront the issue of complexity in the cell—how a small change in one component can affect hundreds of things. The challenge facing the next generation of scientists is to understand cells as systems... how all the components interact and how the interactions define cellular function.
CH: Do you need a really powerful computer to do in silico experiments?
Schilling: No, I just use my laptop—but I'm working on relatively simple problems now using basic linear algebra. In the future, as we try to build models of more complex organisms—such as humans—and apply more intensive mathematical approaches, a supercomputer might be required. We aren't limited by our computational power at the moment, but rather by our lack of biological knowledge and the scarcity of adequate modeling approaches.
CH: What ingredients do you need to perform an in silico metabolism experiment?
Schilling: I start with a list of genes, then I look in textbooks and scientific articles to find out what reactions they catalyze and what's known about them—this provides a "parts catalog" of sorts. Then, I apply simple laws of physics and math principles—basically, describing networks of interacting enzymes as a set of mathematical equations. Out of that comes a prediction of what kinds of products the cell is capable of making under different environmental and genetic conditions.
CH: Will computers replace beakers and petri dishes?
Schilling: No! Much of our knowledge of bacterial metabolism comes from biochemistry research in the 1960s—I rely heavily on this information. In the future, computer models will be used to design experimental programs in research and industry—yet at the same time the experimental results will be required to improve the computer models. So, you could say that they're in a partnership of sorts.
CH: How would you attract young people to study metabolism?
Schilling: I think you have to get people who are interdisciplinary—people who can gain appreciation for all aspects of the problem. It won't work with teams of different specialists just working together on their own subprojects in genetics, biochemistry, or computational biology.
CH: How did you get interested in metabolism and bioengineering research?
Schilling: In my junior year at Duke University, I was a biomedical/electrical engineering double-major and I saw a talk on automated DNA sequencing by Leroy Hood, a genetics researcher at the University of Washington. It was the first time I had seen engineering strategies of any sort being applied to a molecular problem. The next day, I dropped electrical engineering and picked up genetics.
Got It?
Describe one way bacteria become resistant to antibiotics.
What do biological motors use for fuel?
How does an enzyme speed up a biochemical reaction?
What are the three phases of matter?
In 20 years, do you think scientists will still study biology in a laboratory—or will they just do everything on a computer? Give a reason for your answer.