A new understanding of the way enzymes work

This research, both fundamental and applied, was conducted in collaboration with the Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (LCBPT, CNRS/Université Paris Descartes), the Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC, CNRS/Université Paris-Sud 11) and the Laboratoire de Cristallographie et RMN Biologique (LRCB, CNRS/Université Paris Descartes). It was published on the 24 May 2011 on the site of the online journal PLoS Biology.

One of the fundamental principles of the living world is its ability to undergo chemical reactions of great complexity, in an extremely rapid and precise manner, and to sequence them together. It is in this way that cells survive and divide. Specific macromolecules, enzymes in infinitesimal quantities compared to the reactants, catalyze these biochemical reactions and can be reused countless times. But can these proteins essential to life accelerate reactions so efficiently? In fact, the substrate firstly needs to be recognized by the enzyme, come into contact with certain chemical groups specific to it, then undergo a transformation, favored by the chemical environment thereby created and associated with deformations of molecular groups physically close to each other in space. The macromolecular assembly thus attains a highly reactive ephemeral state known as a "transition state," which increases the rapidity of the biochemical reaction by a factor of several hundred billion.

This fundamental knowledge has inspired researchers to identify and improve small compounds that mimic the substrates, without undergoing modifications, but which remain nevertheless fixed for a long time to the enzyme. This persistence in fact makes the macromolecule inactive and incapable of exercising its activity on its natural substrate. Drugs work on exactly the same principle; they usually correspond to inhibitors that mimic the transition state and literally "bond" to the target enzyme, preventing it from operating and inducing associated pathologies. However, the bases of the recognition between the enzyme and its substrate (or inhibitor) remained for a long time poorly understood.

According to the work of the chemist Emil Ficher in 1894 (1), the enzyme recognizes its substrate via a "key-lock" mechanism. It then remained to be understood when and how the so-called "conformational" morphological modifications of the enzyme take place and what processes are involved in these modifications. In 1958, Daniel Koshland proposed a first model (2), which assumes that the small compound firstly interacts with the enzyme and that it is this interaction that induces the conformational change of the macromolecule, thereby making it capable of transforming the substrate. According to this "induced adjustment" model, the substrate thus plays a very active role in the modification of the form of the enzyme. In the alternative "conformational equilibrium" model, formalized in the 1990s (3,4), it is suggested that the enzyme can exist as several -- at least two -- conformational isoforms and that the substrate attaches itself preferentially to one of the minor forms, which is the only one suited for catalysis. In this mechanism, the change of conformation takes place before the reaction and the substrate does not contribute directly to it, unlike the first model.

Over the last few years, numerous data has accumulated in favor of this second mechanism, whereas the first has remained hypothetical, without any tangible experimental proof of its existence. However, the recent work of the team headed by Carmela Giglione Meinnel at the ISV and conducted jointly with Isabelle Artaud of the LCBPT, Michel Desmadril of the IBBMC and Frédéric Dardel of the LRCB has, for the first time, supported the existence of the model proposed by Koshland over 50 years ago. By using a therapeutic target enzyme, the researchers took advantage of the detailed study of a small compound mimicking the substrate, capable of binding very strongly to the enzyme, blocking its activity and thus exhibiting antibiotic, antineoplastic and herbicidal properties. The efficient binding of this compound to the target enzyme, as revealed by a series of structural biological, enzymological and biophysical analyses and computer modeling, requires an "induced adjustment" stage. In other words, it is the small compound which, once bonded to the enzyme, induces its conformational modification.

Thanks to the resolution of the fine structure of this enzyme derived from the chloroplast of the plant Arabidopsis thaliana in several sequential conformational states, the scientists have succeeded in precisely describing the interactions and conformations of each of the partners, enzyme and substrate, at each stage of the reaction. In this respect, the modification of a "pocket," in which one of the groups of the substrate inserts itself, allows the formation of a hydrogen bond, thereby stabilizing the enzyme-substrate complex in the transition state and making it highly reactive to perform the enzymatic hydrolysis reaction efficiently. This study enabled researchers to gather a series of proofs that this model applies to all forms of the enzyme, in particular those found in bacteria, the targets of powerful antibiotics. They have also helped to explain the mechanism by which a therapeutic molecule can bind to its target so that it no longer manages to "unbind" from it, which makes it possible to prolong the effect of the drug beyond the actual treatment.

Consequently, this research work is based on the detailed understanding of a therapeutic mechanism in order to answer a fundamental question and, conversely, this conceptual understanding could, in return, have repercussions in applied biology and in therapeutics. However, as this work demonstrates, the strategies adopted by these extraordinary biological catalyst enzymes are both multiple and plastic, and may also be completed by a conformational selection. These are important factors to take into consideration when designing ab initio or improving the pharmacological properties of drug candidates.