ENZYMOLOGY TODAY

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.