While the catalytic function of enzymes has been well known to researchers for decades, the driving force behind it is still a hotly debated topic. The views on this fundamental issue are condensed around the following two hypotheses: (1) catalytic function originates from preorganized electrostatics; and (2) catalytic function is driven by nonequilibrium dynamical effects. The study gives strong evidence in favor of the first hypothesis. We developed a simple and inexpensive, yet efficient multiscale computational model based on the treatment of the reacting moiety by quantum chemistry, embedded in the enzymatic environment represented by atomic point charges. The approach facilitates investigation of the influence of the enzyme’s electrostatics on the electronic structure of the reacting subsystem, thereby assessing some vital aspects of reactivity and kinetics. Importantly, the approach allows for simple manipulation with the electrostatic environment – the charges can be (selectively or entirely) switched off, scaled, displaced or otherwise modified – giving insight into the role of electrostatics in enzymatic reactions. We applied the above described model to the selected reaction (catalytic step of phenylethylamine oxidation by the monoamine oxidase A enzyme), represented by 100 snapshot structures corresponding to reactants, and 100 snapshot structures corresponding to the transition state. By switching the enzyme’s point charges ON and OFF we demonstrated that electrostatics exhibits decisive catalytic influence by all the considered criteria. Namely, on inclusion of enzyme’s electrostatics the reaction barrier drops by 14 kcal/mol, the charge transfer increases by 40% and the HOMO-LUMO gap pertinent to the reaction decreases by 20%. The catalytic function of the enzyme is rationalized by the stabilizing interaction between the dipole moment of the reacting moiety and the electric field exerted by the charged environment that is noticeably stronger in the transition state than in reactants, thereby lowering the barrier. Our findings support the view that catalysis in enzymes originates from preorganized electrostatics, i.e. enzymes are evolutionary designed in such a way that they, by electrostatic interactions, lower the free energy barrier, resulting in the significantly increased reaction rates.
COBISS.SI-ID: 6556442
Human DNA topoisomerases represent one of the key targets of modern chemotherapy. An emerging group of catalytic inhibitors of human DNA topoisomerase IIslpha comprises a new paradigm directed to circumvent the known limitations of topoisomerase II poisons such as cardiotoxicity and induction of secondary tumors. In our previous studies, 4,6-substituted-1,3,5-triazin-2(1H)-ones were discovered as catalytic inhibitors of topo II alpha. Here, we report the results of our efforts to optimize several properties of the initial chemical series that did not exhibit cytotoxicity on cancer cell lines. Using an optimized synthetic route, a focused chemical library was designed aimed at further functionalizing substituents at the position 4 of the 1,3,5-triazin-2(1H)-one scaffold to enable additional interactions with the topo II alpha ATP binding site. After virtual screening, selected 36 analogues were synthesized and experimentally evaluated for human topo II alpha inhibition. The optimized series displayed improved inhibition of topo IIalpha over the initial series and the catalytic mode of inhibition was confirmed for the selected active compounds. The optimized series also showed cytotoxicity against HepG2 and MCF-7cell lines and did not induce double-strand breaks, thus displaying a mechanism of action that differs from the topo II poisons on the cellular level. The new series represents a new step in the development of the substituted-1,3,5- triazin-2(1H)-one class towards novel anticancer agents.
COBISS.SI-ID: 6615834
In this study substituted 4,5'-bitiazoles, known inhibitors of the bacterial topoisomerase type II DNA gyrase were used as a design starting point. By comparing ATP binding sites of both enzymes, we identified their key differences and similarities that were used in the virtual screening of a targeted chemical library to identify inhibitors that would better bind to the human DNA topoisomerase II? ATP binding site. For selected compounds, we demonstrated at the in vitro level that they act as catalytic ATP-competitive inhibitors of topo II? and bind to its isolated ATPase domain. The compounds were also effective at the cellular level, as some of them exhibited cytotoxicity on the HepG2 and MFC-7 cell lines in the same range as clinically used anticancer drug etoposide. The substituted 4,5'-bitiazoles stopped the cell cycle in the G1 phase, affected the cell proliferation, and did not cause double DNA double-strand breaks, a further evidence that these compounds exhibit a different mode of action at the cellular level compared to topo II poisons.
COBISS.SI-ID: 18365443
We studied dopamine levels in three compartments of the dopaminergic synapse, including the presynaptic neuron cytosol, dopamine storage vesicles, and the synaptic gap. By considering three transport pathways (dopamine transporter (DAT), vesicular transporter (VT), and exocytosis), four simulated scenarios were investigated: homeostasis, application of cocaine, methamphetamine, and reserpine. Recent experiments show that upon cocaine administration, the Drosophila melanogaster DAT permeation rate constant is decreased by 55% and we adopted this value for the human DAT. Amphetamine and methamphetamine block DAT and VT, while reserpine blocks VT; however, their decreased permeation rate constants are not available. A system of three differential equations of dopamine levels as a function of time was developed respectively for the synaptic compartments and was solved numerically. Per computational inference, the cytosol dopamine concentration was noted to increase in the case of methamphetamine and reserpine but was practically unchanged in the case of the cocaine administration. Accordingly, our study suggests that amphetamines and other substances that block VT, but not cocaine or substances that only block DAT, may be etiologically important in the cytosolic dopamine mediation of neurodegeneration in Parkinson disease/ Parkinsonism.
COBISS.SI-ID: 6756378
Nuclear quantum effects, including tunneling, play an important role in the mechanisms of enzyme-catalyzed reactions, with the quantum nature of some atoms, especially the hydrogen involved in the reaction, allowing a faster course of reaction; the kinetic component of tunneling is independent of temperature. The experimental measure of nuclear quantum effects is the H/D kinetic isotope effect, which can be measured relatively easily. The role of nuclear quantum effects was studied by simulation techniques on the example of the enzyme monoamine oxidase A and the substrate phenylethylamine, using two different approaches. In both cases, we were able to predict the value of the kinetic isotope effect very accurately. The influence of nuclear quantum effects (tunneling) on ??the reaction kinetics is relatively small, and a good agreement with the experimental kinetic data confirms that the assumed reaction mechanism is correct, which significantly contributes to the understanding of MAO enzymes.
COBISS.SI-ID: 21912067