The Secret Lives of Enzymes

By Francesco Lanucara, University of Rome La Sapienza

Good news from the cellular yards! Italian scientists from Sapienza Università di Roma unveiled the detailed functioning of one of the most important human enzymes, paving another avenue for the discovery of novel treatments.

Enzymes are proteins operating as essential biological catalysts, which means they promote vital biochemical reactions otherwise unlikely to occur in physiological conditions. Enzymes use raw materials, or reagents, to modify or create new molecules. The so-called cytochrome P-450 is one of the most intriguing family of enzymes currently known. Its key role lies in “oxidative metabolism”, where “oxidative” literally means the use of oxygen as the main reagent. P-450s are characterized by a “catalytic cycle”, that is, the ensemble of all the intermediate forms in which they exist while functioning. Like a machine, they switch from one “state” to another and create intermediates -- with changes in structure (shape), reactivity (what they’re able to do in a precise moment) and reagents (species they’re working with).

“Trapping” and studying such intermediates is an important goal in enzymatic research. What is behind these efforts? Most of the available drugs used to heal many diseases share an action mechanism based on the regulation of P-450 catalytic cycles. Understanding how these intermediates function may offer a unique chance for the design of innovative drugs. When an exogenous compound undergoes a reaction with these P-450 intermediates, its structure is modified such that it is available to be used and afterward disposed of when unnecessary or even toxic. The same reactions could also activate a so-called “prodrug”, i.e. a molecule whose active ingredient is switched on by an enzymatic reaction involving P-450s. This opens the way for the design of new sophisticated molecules whose activation occurs only within the target organ, thus, avoiding the side effects that often limit whether drug therapy works.

The active form of P-450s, known as Compound I, takes part, among other processes, to the “oxidative N-dealkylation” – in order to metabolize exogenous amines, molecules that are commonly present in food and drug preparations. Since Compound I was first discovered, the mechanism of this reaction has been raising a great deal of interest as it is involved in the formation of either the active or toxic form of a drug.

However, the reactions may change depending on the reagent and the involved enzymatic form. For example, the Compound I process is characterized by very reactive and thus unstable intermediates, which are able to change their structure and reactivity so fast that they actually elude any real-time inspection of the process. The whole sequence of events is inferred almost exclusively from the features of the final products. Like a detective looking at the crime scene and trying to re-enact who, where, when and what has been involved in the whole business, researchers have various devices to investigate the secret lives of these enzymes.

Working as a PhD student in my research group in Rome, I took on the challenge to catch these intermediates, exploiting the powerful tool of mass spectrometry (MS). Thanks to technological advances, MS is becoming a fundamental technique in biotechnology and medicinal chemistry. I used a state-of-the-art MS technique, known as electrospray Fourier transform-ion cyclotron resonance (ESI FT-ICR). This experimental method is based on the vaporization of unstable reaction intermediates holding either a positive or negative charge (ions) from the solution to the vacuum, in other words, freeing the ions from all the solvent molecules and any other chemical species. The “gaseous” species are then trapped inside the FT-ICR cell (the reaction vessel), which confines the charged species using magnetic and electric fields and allows them to react with selected substrates. Everything happening inside this “reactor” generates a signal that is monitored and displayed in real time. In essence, the user spies on the whole process following its temporal evolution. In the gas phase, which is a very diluted environment, the otherwise unstable and elusive intermediates, such as the ones involved in the P-450s’ catalytic cycle, can be directly observed and described.

With this approach, I have essentially caught the “thief” in action. By using carefully-designed synthetic models of P-450s, I succeeded in directly observing the intermediates involved in the crucial reactive events of Compound I with amines, thus, mimicking the real process occurring inside living cells. Knowing how enzymes work is the first step in the development of a cure. I believe that this study, along with other results gained by our team, is laying the foundation for a more comprehensive understanding of those biochemical processes whose regulation represent a unique challenge to the treatment of several diseases, such as inflammation and cancer. For now, these results have shed light on the general mechanism and functioning of these enzymes. The importance of these data is recognized by research peers through the publication in one of the top chemistry journals (Journal of the American Chemical Society).

Francesco Lanucara
University of Rome La Sapienza
www.atomiumculture.eu