This collaborative research award in the Chemistry of Life Processes (CLP) program supports work by Prof. Brian Bennett of the Department of Biophysics at the Medical College of Wisconsin to carry out a time-resolved pulsed-electron paramagnetic resonance (EPR) spectroscopic study of the mechanism of iron- and cobalt-containing nitrile hydratases (NHases), in collaboration with Prof. Richard C. Holz (CHE-1058357) at Loyola University, Chicago. The mechanism of the stereospecific hydration of nitriles by NHases is of fundamental interest, and the reaction is of great practical and economic importance in the pharmaceutical industry. Isotopically labeled (H-2, C-13, N-15) substrates and NHase enzyme will be incubated and collected by rapid-freeze-quench to trap pre-steady-state and steady-state intermediates, and these will be interrogated by pulsed EPR methods. Simulations will be used to obtain spin-Hamiltonian parameters from which the structures of the intermediates can be elucidated, and a mechanism deduced.
It is anticipated that this research will pave the way for the design of chemical catalysts and engineered enzymes whose properties can be tailored toward specific substrates and desired reaction properties. This will greatly increase the range of pharmaceuticals and intermediates that rely on stereoselective nitrile hydration that can be profitably brought to market, and stimulate further research into new compounds of this class.
This project is part of a collaborative research project to study the mechanism of an enzyme known as nitrile hydratase. Nitrile hydratase is a biological catalyst that hydrates (adds water to) nitriles (cyanides), forming amides. Many important pharmaceuticals (e.g. penicillin, LSD) are amides. and their synthesis often involves the hydration of the corresponding nitrile. Many of these amides are chiral molecules, possessing "left-handed" and "right-handed" forms (optical isomers) that can appear chemically identical and are produced in a 50:50 ratio during chemical synthesis. Unfortunately, as with, for example, the morning sickness drug thalidomide, the two forms may have very different biological activities and effects on the human body, and they are often extremely difficult to separate from one another. A method for synthesizing chiral amides of only one optically isomeric form is therefore highly desirable, and the use of nitrile hydratase, in contrast to chemical methods, is a potentially convenient method. In addition, nitrile hydratase operates at ambient temperatures and pressures, and in water-based solutions or immobilized in gels rather than at high temperatures and pressures and in organic solvents. Therefore, the effects of its use on the environment are extremely benign compared to chemical synthesis or catalysis. Indeed, as nitriles themselves are a common environmental toxin (as pesticides), nitrile hydratase also has a role in reclamation of contaminated land. Thus, nitrile hydratase has roles in pharmaceutical chemistry, "green" chemistry (e.g. in the industrial synthesis of nicotinamide), and environmental chemistry. In order to exploit the catalytic potential of nitrile hydratse for a wide variety of precursor molecules and ultimate products, and to maximize efficiency and specificity of the reactions, we need to tailor the properties of nitrile hydratase catalysis through protein engineering or synthesis of chemical catalysts with nitrile hydratse-mimicking chemistry ("biomimetic" catalysts). To be able to engineer precise ntrile-hydrating chemical behavior into a molecule, we first need to find out the mechanism of action of nitrile hydratase, i.e. how it does what it does and what structural factors are important in determining how it does what it does. This is the goal of this collaborative research project, that brings a number of investigators together to work on this one problem using multiple techniques and approaches. Two of the most important questions are, what is the source of the water molecule that hydrates the nitrile and how is it made more reactive toward the nitrile? There are two "flavors", closely related, of nitrile hydratase, one of whcih contains an atom of the metal iron and the other that contains a cobalt atom. These metals are bound in essentially the same very unusual manner that suggests that they are vitally important to the function of the enzyme. Using a technique known as electron-nuclear double resonance spectroscopy (ENDOR), we identified a hydrogen atom very close to the metal center that suggested a water molecule bound to the metal ion and was a good candidate for the reactive water (the "nucleophile"). By mixing the enzyme and a nitrile for a very short time (1/100th of a second) and freezing the sample, we were able to trap an intermediate in the reaction that would correspond to the transfer of the water to the nitrile. We used a technique, electron paramagnetic resonance (EPR), to obtain a "fingerprint" of this species. With a non-reactive nitrile analog we were then able to recreate the fingerprint species that, unlike the real catalytic intermediate, was sufficiently stable to allow crystals to be grown. X-ray analysis of the crystals provided a picture of the active site that showed that the activated water nucleophile was not actually provided by the metal ion, but by one of the chemical groups that bind the metal (a cysteine-sulfenic acid ligand). Presumably by ionizing (making negaitively charged) the water molecule to a hydroxyl, the sulfenic acid group makes it much more reactive toward the nitrile. Therefore, we have ascertained the source of the reactive water molecule and a likely mechanism of its activation. Another outstanding mechanistic question was, how does the nitrile substrate bind to the enzyme? This is important in determining which optical isomer is formed. To answer this, we applied a technique known as electron-spin-echo envelope-modulation (ESEEM) that is particularly sensitive to the number of nitrogen atoms in the vicinity of the metal ion. Upon examination of trapped intermediates by ESEEM we observed an increase in the number of nitrogens bound to iron. The only possible source of this was the substrate nitrile itself. Thus, we have determined that the nitrile binds to the metal via the nitrile nitrogen atom, and a water molecule is bound to and activated by a metal-binding sulfenic acid group and reacts with the adjacent metal-bound nitrile. This represents substantial progress in determining the mechanism of nitrile hydratase and paves the way for the design of tailored catalysts.
