Enzymology's New Frontiers

Investigators are learning about structures and rethinking old theories | By Mark Greener

"There's no big mystery to being an enzymologist. All you have to have is a razor blade and a liver."

--Gordon Tomkins to Julius Axelrod, circa 1950

Courtesy of International Union of Biochemistry and Molecular Biology
METABOLIC CARTOGRAPHY: A portion of the 22nd edition of Sigma-Aldrich's Metabolic Pathways map which was designed by Donald E. Nicholson at the University of Leeds. The enzymes are indicated by IUBMB Commission numbers. For example, in the top, left-hand corner [above UDP] is 2.4.99.7, which is a sialyltransferase.

Today's researchers rarely resort to razor blades to understand how enzymes function. Investigators can capture the kinetics of a single enzyme molecule or use site-directed mutagenesis and crystallography to appreciate the subtleties of enzymatic action.

Such technological advances help enzymologists understand the fundamental biological processes that have an impact on life sciences as diverse as systems biology, neurology, and ecology. Modern enzymology also has numerous commercial applications, which include a plethora of new pharmaceutical targets.

The field also has an abundance of unsolved, pressing problems: many of the current frontiers in enzymology require multidisciplinary approaches. In particular, the field needs to evolve away from examining enzymes in isolation. Increasingly, enzym-ology aims to understand entire biochemical pathways and the way in which genotypic and proteomic variations influence outcomes.

"There is no doubt in many people's minds that there is, or will be, a renaissance in enzymology as a discipline," says Hong Qian, associate professor at the Department of Applied Mathematics, University of Washington, Seattle. "But it will study many enzymes in a complex biochemical reaction network, not just one enzyme at a time." As a result, many enzymologists are moving away from relatively simplistic deterministic kinetics and toward sophisticated, probabilistic mathematics and nonequilibrium thermodynamics.

A recent study by research fellow Kirsten Donald, Department of Zoology, University of Otago in New Zealand, and colleagues¹ underscores the power of merging traditional enzymology and new technology in the study of biological systems. The investigators combined quantified gene expression using fluorogenic real-time PCR and classic enzymatic assays to understand the role and regulation of proteolysis in the early development of the Pacific oyster Crassostrea gigas. "Measuring both expression and activity over time allowed us to understand how protein-turnover enzymes were regulated," she remarks. Some enzymes, such as leucine aminopeptidase, are regulated at the transcriptional level. Others, including cathepsin B, undergo posttranscriptional modification.

UNDERSTANDING STRUCTURES Full structural analysis of the enzymes in the pathway is another landmark of the systems biology frontier and a multidisciplinary endeavor.² Indeed, recent studies using site-directed mutagenesis and crystallography have shown researchers the finer details of enzymatic action. Thomas Dandekar, from the European Molecular Biology Laboratory in Heidelberg and the Department of Bioinformatics at the University of Würzburg, Germany, suggests that such findings could help inform "nearly all biological disciplines" including microbiology, immunology, neurobiology, and ecology.

Crossing this frontier means moving beyond genomics; sequence homology may not offer an appropriate surrogate for structure. Even proteins with more than 80% homology can show different structural features in the active domain that may influence specificity, selectivity, and reactivity.³ Against this background, crystallography becomes increasingly important. "We have learned so much about how enzymes work by the analysis of their structures," says professor Gail Johnson, Department of Psychiatry at the University of Alabama, Birmingham.

By 2000, researchers had structurally characterized some 640 enzymes, including, for example, the enzymes involved in the glycolytic pathway.³ Since then, the number of characterized structures has tripled. The BRENDA database (http://www.brenda.uni-koeln.de/), maintained by the University of Cologne, Germany, collates data on the 35,000 or so enzymes classified according to the Enzyme Commission nomenclature. The database now contains some 1,900 crystallography results as well as the tertiary structures of 11,000 enzymes.

However, the number of structural characterizations should increase markedly over the next few years. Traditionally, determining a protein's 3-D structure using X-ray crystallography took months or even years. But technological advances that integrate robotic systems and new software for data collection and processing now produce fully automated, high-throughput crystallography systems, which can determine the structures of 18 crystals of aldolase and 54 crystals of protein tyrosine phosphatase 1B in 42 and 80 hours, respectively.²

This growing structural database holds some surprises. For instance, researchers recognized years ago that tissue transglutaminase bound and hydrolyzed guanine triphosphate (GTP). But no domain showed homology with any other known G protein. Indeed, mutational studies suggested that the GTP binding and hydrolysis domain resided in the N-terminus. In 2002, crystallography of tissue transglutaminase revealed a unique GTP binding and hydrolysis domain in the C-terminal.⁴ As Johnson remarks, "You never know until you see the structure!"

Site-directed mutagenesis augments crystallography's revelations. For example, it allows researchers to substitute cysteine with serine. This retains the residue's size and chemical character and, therefore, does not disrupt protein folding. Few such substitutions lead to a total loss of activity. "This suggests that active sites of enzymes are optimized for the catalytic step and promote catalysis even when the key residue is knocked out," comments Wilbur Campbell, emeritus professor of biochemistry at Michigan Technological University, Houghton.

RETHINKING THE THEORIES Enzymologists working on their discipline's new leading edge need to make a "conceptual shift."⁵ Enzyme systems do not reach equilibrium even when they seem stationary; the system is in constant flux. "Enzymologists have not paid enough attention to this, but metabolic engineers emphasized the flux in biochemical reactions for years," Qian notes. Characterizing enzymes and their reactions in living biochemical networks, he argues, means moving beyond usual equilibrium physical chemistry and kinetics to include a branch of physics called nonequilibrium thermodynamics. "This is the theoretical foundation of the new enzymology," he says.

Traditional enzymology experiments aim to characterize, for example, V_max (maximum velocity) and K_m, to help define the enzyme's kinetic behavior. K_m offers a rough measure of how much substrate is needed to fuel V_max. But these parameters are often either unknown or extrapolated from different organisms or species. "Computing results from such a model is not going to be very satisfying," Qian says. So, to produce such satisfying results, researchers are examining a number of alternative mathematical approaches, including stochastic modeling.

An enzyme molecule constantly collides with the surrounding solvent molecules. As a result, the enzyme's thermodynamics fluctuate rapidly. Traditionally, researchers regarded these fluctuations as experimental noise, which could be ignored; the bulk measurements of steady-state enzyme assays masked such thermodynamic subtleties. But Qian notes in a recent paper⁶ that such thermodynamic fluctuations could contain valuable data, allowing enzyme kinetics to be modeled. This means, however, shifting from the deterministic kinetics of molecular concentrations in a traditional enzymatic assay to a stochastic model, based on the probability that a molecule will be at a particular state.

Traditional enzymatic assays are at steady state. The researcher takes bulk measurements of the turnover of large amounts of substrate molecules by multiple enzyme molecules. But increasingly, enzymologists capture the kinetics of a single enzyme molecule using a variety of methods, such as confocal fluorescence microscopy and fluorescence correlation spectroscopy.

So, enzymologists ask stochastic rather than deterministic questions, such as, "What is the probability of the enzyme being in an active configuration at a particular time?" A stochastic approach illuminated, for example, ATPase kinetics in motor protein movements. "Considering that the number of enzyme molecules inside a cell can often be as low as a few copies, there is a great need to think of enzymological reactions as stochastic processes in the same way that physiologists study membrane channels," adds Qian.

RIBOZYMES OPEN NEW VISTAS Meanwhile, scientists' growing recognition of the importance of nonprotein enzymatic actions has opened another new vista for enzymology. Victoria DeRose, associate professor of chemistry at Texas A&M University, College Station, and author of a recent review of two decades' worth of research into ribozymes,⁶ remarks that enzymologists formulated ribozymes into enzyme-substrate constructs that could be analyzed using Michaelis-Menten kinetics. This allowed researchers to compare directly the catalytic activity of ribozymes and protein enzymes. "The power of enzymology as a discipline is apparent in the way that ribozymes have been examined since the discovery of catalytic RNA," says DeRose.

Click to view enlarged glycolytic pathway diagram (38K)

Researchers had "immediate interest in determining how, and how well, RNA could perform the standard 'tricks' of enzymes, such as positioning reactants and lowering transition state barriers," she continues. "The new vista for enzymology is to understand the mechanics by which RNA performs the roles of a biological catalyst."

DeRose believes that electrostatics may be one of the most important of the several structural differences between ribozymes and proteins. "Internal electrostatics can play an important role in catalysis, such as by altering the acidity of a reactive group or neutralizing charges during a reaction," she says. "RNA has a negatively charged phosphodiester backbone with no natural positive charges, requiring a counter-ion atmosphere. In comparison with proteins, this is an exotic ionic environment."

Flexibility represents another important frontier in ribozymology. A growing and compelling body of evidence, including recent single-molecule studies, suggests that structural changes influence ribozyme function.^7,8 "It is important to determine the nature of these changes, and to account for them in mechanisms based on kinetic measurements," DeRose comments. "Ribozymes lend themselves well to single-molecule fluorescence experiments because they are relatively easy to modify and because they can have large-scale movements that give clear fluorescence signals."

COMMERCIAL BENEFITS Meanwhile, some companies find that advances in enzymology help hone their competitive edge. "One of the most important advances in applied enzymology recently has been the more general acceptance by the chemical industry that enzymes can achieve things that are impossible using traditional chemical catalytic methods," says biotechnology professor Chris Bucke, University of Westminster, London. (The cell factory discussed below offers one example of this.)

Enzymes also provide a tempting and lucrative target for pharmaceutical companies. "Most therapeutics mediate their desired effect and unwanted side effects through enzymes," remarks Gunter Fischer, director of the Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg, Germany. Indeed, blockbuster drugs such as angiotensin-converting enzyme (ACE) inhibitors, statins, and several AIDS drugs act by inhibiting key enzymes. ACE inhibitors, for example, target a key enzyme in the renin-angiotensin system, which plays a central role in controlling blood pressure. Statins inhibit the rate-limiting step in cholesterol biosynthesis (an enzyme called HMG-CoA reductase), thereby reducing blood lipid levels. Several AIDS drugs target key enzymes in the HIV life cycle.

Researchers continue to target enzymes in their search for more effective medications for common diseases. Approximately 200 million people contract malaria each year; perhaps two million die because of it. The malaria parasite, Plasmodium falciparum, increasingly resists conventional medications. P. falciparum invades erythrocytes, destroys as much as 80% of the hemoglobin, and then feeds on the degraded hemoglobin. Two enzymes (plasmepsins I and II) are critical in driving the hemoglobin's destruction. Researchers have identified several compounds that block these enzymes, raising the prospect of new treatments.

The two plasmepsins belong to a large enzyme family called aspartic peptidases. Other enzymes in this family have links to several diseases, including renin (hypertension), cathepsin D (breast cancer metastasis), b-secretase (Alzheimer disease), HIV-1 peptidase (AIDS) and aspartic peptidases secreted by candidal infections.⁹ Drugs acting on aspartic peptidases, such as the ACE inhibitors, are available now; others are in development.

Insulin as well as many growth factors and cytokines act by binding to specific receptors associated with enzymes, usually a kinase. When the ligand binds, the kinase phosphorylates certain amino acids that make up the receptor protein. This autophosphorylation allows the receptor to interact with other enzymes inside the cell, thereby amplifying the signal.

Mutated proteins in signaling pathways controlled by kinase-linked receptors probably contribute to several cancers. For example, signaling linked to the epidermal growth factor receptor (EGFR) contributes to cell proliferation, apoptosis, angiogenesis, and metastasis. Many drugs targeting this and other kinase-linked signaling pathways are in development or are marketed in some parts of the world. Indeed, Iressa (gefitinib), which targets EGFR, is approved in the United States for the third-line treatment of non-small-cell lung cancer resistant to other chemotherapies.¹⁰

Despite these successes, drug companies might not fully exploit enzymology's potential. "To fully realize the therapeutic potential of enzymes, a more knowledge-based approach, rather than high-throughput screening, would be desirable," Max Planck's Fischer says. "Enzymologists can provide this knowledge by developing a more detailed theory of how enzyme- ligand [interactions] and enzyme catalysis is accomplished."

The pharmaceutical industry, Dandekar says, focuses too much on in vitro or cellular systems, which may be a reduction too far. "Often you get nasty surprises at the end of the pharmacological pipeline from side-effects," he says. "We need more top-down approaches, even if that is difficult." Alabama's Johnson adds that sometimes the pharmaceutical sector may jump the gun by targeting specific enzymes. "In some cases, they try to develop specific inhibitors before it is well established that enzyme X is actually involved in the pathogenic process."

Enzymology also affects mundane aspects of daily life, and there are few activities more forgettable than doing the laundry. Subtilisin, which enzymatically degrades protein and is used in laundry powders, is the most highly engineered enzyme.¹¹ Subtilisin must remain active at the high temperatures inside washing machines. Campbell, who is also president of the Nitrate Elimination Company, Lake Linden, Mich., a biotech organization that produces nitrate reductase and other enzymes for commercial purposes, notes that comparing the structural characteristics of thermophilic enzymes to those that function at lower temperatures may reveal the properties that make the former thermally stable and, therefore, more appropriate for commercial applications.

Westminster's Bucke, an applied enzymologist who divides his time between industry and academia, adds that site-directed mutagenesis improves industrial enzymes used to make glucose and fructose syrups. "Native *-amylase, which is used to start the hydrolysis of starch, is astonishingly temperature-stable and is used at more than 100°C, but requires calcium ions to retain that stability," he explains. "Site-directed mutagenesis produced an equally stable enzyme that no longer requires added calcium. This results in savings in downstream processing."

The cell factory is another recent innovation that uses genetically engineered organisms (such as Escherichia coli) to synthesize particular proteins. There's nothing new in that, of course. But enzymologists can engineer the pathway used in the cell factory to make the protein and eliminate unwanted reactions and control mechanisms. This means that the organism produces higher protein yields with fewer side-products than older genetically engineered organisms. Bucke cites the example of a cell factory developed by Genencor in Quebec, Tate & Lyle in London, and Dupont that yields propan-1,3-diol, which is used to generate propylene terephthalate at "cents per pound."

Courtesy of National Park Service

THE DARNDEST PLACES: Scientists isolated the thermostable DNA polymerase Taq, an enzyme that drives PCR, from Thermus aquaticus Yellowstone type-1, a resident of geysers like this one at Yellowstone National Park.

TACKLING THE FUTURE Elena Ghibaudi, research assistant at the Department of Inorganic, Physical, and Material Chemistry, University of Torino, Italy, argues that enzymologists need to engage in improved dialogue and information exchanges with researchers in other life sciences. She notes that universities tend to train scientists to focus their attention on specific aspects of a discipline; they rarely offer a general picture. "People are trained to face scientific problems from a relatively restricted perspective," says Ghibaudi.

On the one hand, this restricted perspective encourages life scientists to develop specific competencies. On the other, improved dialogue, information exchanges between disciplines, and a cooperative approach could solve some fundamental problems, says Ghibaudi. "Notwithstanding the great number of conferences organized by life scientists all over the world, I have the impression that not enough real opportunities for discussion and confrontation between disciplines are created." She calls for a multidisciplinary approach, stimulated in part by systems biology. "By dealing with cell complexity, systems biology implies multidisciplinarity and a continuous confrontation between viewpoints that are often far from each other," she says.

Speaking about complexity, Dandekar considers the discovery of how enzymes work to be the most pressing problem facing investigators. Four main research areas predominate: 1) determining how enzymes achieve transition state catalysis; 2) determining the protein sequence that leads to a particular enzymatic activity or useful structure, known as reverse folding; 3) characterizing how environmental factors and allosteric interactions modify enzyme action; and 4) gaining a better understanding of protein folding.

Courtesy of V.J. DeRose, Chem Biol, 9:961:69, 2002.

METALS AND BASES:Possessing fewer reactive groups than do amino acids, ribozymes seem to do their jobs using little more than metal ions and nucleotide bases with potentially altered pKa's. Shown here are proposed reaction mechanisms for three, well-characterized ribozymes.

An additional challenge, says Fischer, lies in developing reliable techniques for predicting protein- ligand interactions or protein structures. For example, homology modeling predicts protein structure from the primary amino acid sequence, but the approach is restricted to proteins that have similar codes to known structures. "We need a method to predict structures and protein-ligand interactions ab initio," he comments. Currently, poor understanding of the basic physical and chemical principles and informatic limitations hinder this approach.

For Michigan Tech's Campbell, "The most pressing question in protein chemistry is: What are the rules for the 3-D folding of a protein? ... [Researchers] need to know how to design a new enzyme so that it folds to a stable structure; then we can focus on active-site design so that new enzymes can be created. We then need to understand how we can improve the stability of existing enzymes to make more robust catalysts that will be more useful in commercial applications. Once we can design highly stable proteins with new and improved catalytic activity, enzymology will be a mature science."

All this without a razor blade in sight, save for the one forged by 14th century philosopher William of Occam.

Mark Greener (biowriter@markgreener.fsnet.co.uk) is a freelance bioscience writer in Cambridge, UK.

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