Changes in dynamics of α-chymotrypsin due to covalent inhibitors investigated by elastic incoherent neutron scattering

Abstract

An essential role of enzymes is to catalyze various chemical reactions in the human body and inhibition of the enzymatic activity by small molecules is the mechanism of action of many drugs or tool compounds used to study biological processes. Here, we investigate the effect on the dynamics of the serine protease α-chymotrypsin when in complex with two different covalently bound inhibitors using elastic incoherent neutron scattering. The results show that the inhibited enzyme displays enhanced dynamics compared to the free form. The difference was prominent at higher temperatures (240-310 K) and the type of motions that differ include both small amplitude motions, such as hydrogen atom rotations around a methyl group, and large amplitude motions, such as amino acid side chain movements. The measurements were analyzed with multivariate methods in addition to the standard univariate methods, allowing for a more in-depth analysis of the types of motions that differ between the two forms. The binding strength of an inhibitor is linked to the changes in dynamics occurring during the inhibitor-enzyme binding event and thus these results may aid in the deconvolution of this fundamental event and in the design of new inhibitors.

Influence of Enantiomeric Inhibitors on the Dynamics of Acetylcholinesterase Measured by Elastic Incoherent Neutron Scattering

“…This shift in the scores is visible in all PC: s but mostly in that PC2 is dominated by the cubic water phase transition at 240 K, primarily apparent in the free m AChE and m AChE-568R samples. This event is clearly associated with the Q : s around 1.59–1.81 Å –1 (demonstrated by their high loadings in Figure B) and matches with the values for ice Bragg peaks (see the Supporting Information, Table S3) and have been reported before. , To summarize the PCA for the IN6 measurements, the largest variation in the data was the temperature trend, the second largest represents the ice Bragg reflections around 1.7 Å –1 and the melting of water, and the third largest showed additional subtle difference between free and inhibited m AChE.…”

“…Huperzine A has been shown to not affect or possibly increase , the dynamics of human AChE ( h AChE), whereas the covalent inhibitor soman decreased its dynamics . The same ligand-dependent trend has been observed for other protein–inhibitor complexes using nuclear magnetic resonance (NMR) or neutron scattering, where complex formation led to increased − or decreased dynamics. , …”

“…To evaluate the EINS measurements of the AChE dynamics on the pico- to nanosecond time scale, the classical univariate analysis methods such as the sum of the intensities and the mean-square displacement (MSD) and a newly developed model to derive the mean-square position fluctuations (MSPFs) were used. In addition to these methods, we have recently presented and used multivariate methods including principal component analysis (PCA) , and orthogonal partial least-squares discriminant analysis (OPLS-DA) , to analyze the EINS data . Here, the univariate and multivariate methods complemented each other and allowed for a detailed analysis of all available scattering angles (or momentum transfers), leading to statistically sound conclusions.…”

Dynamics of proteins in solution

“…Andersson et al . (2017) investigated the effect of two different inhibitors, namely TPCK and chymostatin, on the dynamics of the serine protease α -chymotrypsin. From the analysis of the EINS signal, the authors concluded that the inhibited enzymes underwent a dynamical transition at lower temperatures and in a more cooperative way leading to bigger amplitudes of motions at higher temperatures (up to 310 K).…”

“…From the analysis of the EINS signal, the authors concluded that the inhibited enzymes underwent a dynamical transition at lower temperatures and in a more cooperative way leading to bigger amplitudes of motions at higher temperatures (up to 310 K). Andersson and collaborators deduced that the inhibitor either directly allows for larger amplitudes of the enzyme motions, or influences the water network around the enzymes in a way that permits more degrees of motional freedom leading to a lowering of the potential energy barrier seen by the enzyme atoms (Andersson et al ., 2017).…”