Fundamental questions still remain regarding the detailed mechanisms of enzyme activity and its relationship to enzyme structure. The two most powerful tools that have been brought to bear on these questions in modern times are the continued development and use of biophysical probes of protein structure, and the application of molecular biological methods to enzymology. X-ray crystallography continues to be used routinely to solve the structures of enzymes and of enzyme—ligand complexes. In addition, new NMR methods and magnetization transfer methods make possible the assessment of the three-dimensional structures of small enzymes in solution, and the structure of ligands bound to enzymes, respectively.

The application of Laue diffraction with synchrotron radiation sources holds the promise of allowing scientists to determine the structures of reaction intermediates during enzyme turnover, hence to develop detailed pictures of the individual steps in enzyme catalysis. Other biophysical methods, such as optical (e.g., circular dichroism, UV—visible, fluorescence) and vibrational (e.g.,infrared, Raman) spectroscopies, have likewise been applied to questions of enzyme structure and reactivity in solution. Technical advances in many of these spectroscopic methods have made them extremely powerful and accessible tools for the enzymologist. Furthermore, the tools of molecular biology have allowed scientists to clone and express enzymes in foreign host organisms with great efficiency. Enzymes that had never before been isolated have been identified and characterized by molecular cloning. Overexpression of enzymes in prokaryotic hosts has allowed the purification and characterization of enzymes that are available only in minute amounts from their natural sources. This has been a tremendous advance for protein science in general.

The tools of molecular biology also allow investigators to manipulate the amino acid sequence of an enzyme at will. The use of site-directed mutagenesis (in which one amino acid residue is substituted for another) and deletional mutagenesis (in which sections of the polypeptide chain of a protein are eliminated) have allowed enzymologists to pinpoint the chemical groups that participate in ligand binding and in specific chemical steps during enzyme catalysis.

The study of enzymes remains of great importance to the scientific community and to society in general. We continue to utilize enzymes in many industrial applications. Moreover enzymes are still in use in their traditional roles in food and beverage manufacturing. In modern times, the role of enzymes in consumer products and in chemical manufacturing has expanded greatly. Enzymes are used today in such varied applications as stereospecific chemical synthesis, laundry detergents, and cleaning kits for contact lenses.

Perhaps one of the most exciting fields of modern enzymology is the application of enzyme inhibitors as drugs in human and veterinary medicine. Many of the drugs that are commonly used today function by inhibiting specific enzymes that are associated with the disease process. Aspirin, for example, one of the most widely used drugs in the world, elicits its antiinflammatory efficacy by acting as an inhibitor of the enzyme prostaglandin synthase. As illustrated in Table 1.1, enzymes take part in a wide range of human pathophysiologies, and many specific enzyme inhibitors have been
developed to combat their activities, thus acting as therapeutic agents. Several
of the inhibitors listed in Table 1.1 are the result of the combined use of biophysical methods for assessing enzyme structure and classical pharmacology
in what is commonly referred to as rational or structure-based drug design.
This approach uses the structural information obtained from x-ray crystallography
or NMR spectroscopy to determine the topology of the enzyme active site. Next, model building is performed to design molecules that would fit well into this active site pocket. These molecules are then synthesized and tested as inhibitors. Several iterations of this procedure often lead to extremely potent inhibitors of the target enzyme.

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The oldest known reference to the commercial use of enzymes comes from a description of wine making in the Codex of Hammurabi (ancient Babylon,circa 2100 ..). The use of microorganisms as enzyme sources for fermentation was widespread among ancient people. References to these processes can be found in writings not only from Babylon but also from the early civilizations of Rome, Greece, Egypt, China, India. Ancient texts also contain a number of references to the related process of vinegar production, which is based on the
enzymatic conversion of alcohol to acetic acid. Vinegar, it appears, was a common staple of ancient life, being used not only for food storage and preparation but also for medicinal purposes.

Dairy products were another important food source in ancient societies. Because in those days fresh milk could not be stored for any reasonable length of time, the conversion of milk to cheese became a vital part of food production, making it possible for the farmer to bring his product to distant markets in an acceptable form. Cheese is prepared by curdling milk via the action of any of a number of enzymes. The substances most commonly used
for this purpose in ancient times were ficin, obtained as an extract from fig trees, and rennin, as rennet, an extract of the lining of the fourth stomach of a multiple-stomach animal, such as a cow. A reference to the enzymatic activity of ficin can, in fact, be found in Homer’s classic, the Iliad:

As the juice of the fig tree curdles milk, and thickens it in a moment though it be liquid, even so instantly did Paee¨ on cure fierce Mars.

The philosopher Aristotle likewise wrote several times about the process of milk curdling and offered the following hypothesis for the action of rennet:

Rennet is a sort of milk; it is formed in the stomach of young animals while still being suckled. Rennet is thus milk which contains fire, which comes from the heat of the animal while the milk is undergoing concoction.

Another food staple throughout the ages is bread. The leavening of bread by yeast, which results from the enzymatic production of carbon dioxide, was well known and widely used in ancient times. The importance of this process to ancient society can hardly be overstated.

Meat tenderizing is another enzyme-based process that has been used since antiquity. Inhabitants of many Pacific islands have known for centuries that the juice of the papaya fruit will soften even the toughest meats. The active enzyme in this plant extract is a protease known as papain, which is used even today in commercial meat tenderizers. When the British Navy began exploring the Pacific islands in the 1700s, they encountered the use of the papaya fruit as a meat tenderizer and as a treatment for ringworm. Reports of these native uses of the papaya sparked a great deal of interest in eighteenth-century Europe, and may, in part, have led to some of the more systematic studies of digestive enzymes that ensued soon after.

From: Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
Robert A. Copeland

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Life depends on a well-orchestrated series of chemical reactions. Many of these reactions, however, proceed too slowly on their own to sustain life. Hence nature has designed catalysts, which we now refer to as enzymes, to greatly accelerate the rates of these chemical reactions. The catalytic power of enzymes facilitates life processes in essentially all life-forms from viruses to man. Many enzymes retain their catalytic potential after extraction from the living organism, and it did not take long for mankind to recognize and exploit the catalytic power of enzyme for commercial purposes. In fact, the earliest known references to enzymes are from ancient texts dealing with the manufacture of cheeses, breads, and alcoholic beverages, and for the tenderizing of meats.

Today enzymes continue to play key roles in many food and beverage manufacturing processes and are ingredients in numerous consumer products, such as laundry detergents (which dissolve protein-based stains with the help of proteolytic enzymes). Enzymes are also of fundamental interest in the health sciences, since many disease processes can be linked to the aberrant activities of one or a few enzymes. Hence, much of modern pharmaceutical research is based on the search for potent and specific inhibitors of these enzymes.

The study of enzymes and the action of enzymes has thus fascinated scientists since the dawn of history, not only to satisfy erudite interest but also because of the utility of such knowledge for many practical needs of society. This brief chapter sets the stage for our studies of these remarkable catalysts by providing a historic background of the development of enzymology as a science. We shall see that while enzymes are today the focus of basic academic research, much of the early history of enzymology is linked to the practical application of enzyme activity in industry.

From: Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
Robert A. Copeland

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