The mechanisms of reaction of chorismate mutase enzymes, as well as chalcone isomerase, and the hyperthermophilic glycerol phosphate isomerase will be discussed. The mechanisms of these single substrate enzymes do not involve covalent intermediates and the rate constant for enzymatic reaction (kcat) and the uncatalyzed reaction in water (ko) are both first order. The efficiency of catalysis by an enzyme is, by convention, equal to (kcat/ko) at pH 7.0. Refering to eq. 1, these features simplify the quest to understand the roles in determining enzyme efficiency in the difference of water and enzyme stabilization of NAC and TS. [The present use of the term TS stabilization is an adduct to Pauling's suggestion of TS recognition by the addition of nucleopilic, general-base, general-acid catalysis, the release of solvent and release of strain on E·S E·TS etc].
Our tools have been molecular dynamic (MD) simulations, thermodynamic integrations (TI) and two-dimensional SCCDFTB/MM and TIP3P/MM. Both MD+TI and QM/MM results show that 85 - 90% of the advantage of the chorismate mutase reaction is due to the ability of the enzyme to stabilize NAC. Transition state stabilization is of very little importance as shown by essentially the same TS structures and charge distributions in gas phase, water and enzyme as well as near equal charge densities in enzyme bound NAC and TS.
Chalcone isomerase catalyzes a ring formation in the conversion of chalcone to S-flavanone. E. coli chorismate mutase and chalcone isomerase reactions share identical Km and kcat values. The chalcone isomerase reaction is driven by release of strain in the E·S complex, the release of three water molecules, and general acid catalysis by Lys109-NH3+ on E·S E·TS. These features are summed as TS stabilization.
How do thermophilic enzymes work? Long-term MD simulations were carried out with a indole glycerol synthase at various temperatures from 25° C to 110° C and mole fractions of E·S present as E·NAC determined. A change of 4,000 fold in the rate constant is accompanied by a change of 1,000 fold in NAC population. The effect of temperature is a ground state phenomenon. Examination of E·S structures with temperature reveal that the very favorable structure at 110° becomes distorted gradually on lowering the temperature. This is shown to be the result of electrostatic bonds becoming shorter as temperatures drops and distortion of the active site results. We propose this as, probably, a general explanation for the temperature dependence of reaction rates of thermophilic enzymes.
The tight coupling between chemical events and conformational properties is believed to be crucial to the function of many biological systems. The precise mechanism behind the coupling, however, is often poorly understood and difficult to probe based on experiments alone. Our group develops and applies powerful molecular simulation techniques to explore such type of coupling in the context of catalysis, signaling and bioenergy transduction. Several recent examples will be discussed: the possible role of enzyme dynamics in the catalysis of cyclophilin A will be analyzed in connection with recent NMR studies; preliminary results regarding the mechanochemical coupling in the biomolecular motor, myosin-II, will also be presented.
Theoretical studies of the impact of enzyme motion on proton, hydride, and proton-coupled electron transfer reactions in enzymes will be presented. The quantum mechanical effects of the active electrons and transferring proton are included in the calculations. The investigation of proton-coupled electron transfer in the enzyme lipoxygenase will be discussed . The experimentally measured deuterium kinetic isotope effect of 80 at room temperature is found to arise from the small overlap of the reactant and product proton vibrational wavefunctions in this nonadiabatic reaction. The calculations illustrate that the proton donor-acceptor vibrational motion plays a vital role in the proton-coupled electron transfer reaction. The study of hydride transfer in the enzyme dihydrofolate reductase (DHFR) will also be discussed [2-4]. An analysis of the simulations leads to the identification and characterization of a network of coupled motions that extends throughout the enzyme and represents equilibrium conformational changes that facilitate the charge transfer process. Mutations distal to the active site are shown to significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and thereby changing the probability of sampling conformations conducive to the catalyzed reaction [3,4].
Linking protein motions to the efficiency of enzymatic bond making/breaking processes presents a formidable experimental challenge. For a complete picture, it will be important to define both the time scales of particular motions (in relation to the chemical event itself) and the structural units within a protein whose motions correlate with catalysis. In this context, my laboratory has focused on the cleavage of C-H bonds, whose reactions almost uniformly show a large tunneling component. Using a modified Marcus picture to formulate the H-transfer event, it is possible to parse the process into three terms that include: (i) the Frank-Condon wave function overlap (for the donor and acceptor C-H bonds), (ii) the environmental reorganization and driving force that accompany the hydrogen transfer, and (iii) a gating term that describes the degree to which the donor and acceptor atom distance must change to achieve effective wave function overlap. With these three terms, it is possible to explain a wide range of behaviors in divergent protein systems. This talk will illustrate the insights we have learned using several illustrative enzyme systems.
This seminar will describe ongoing research on the nature of chemical reactions in enzymes. Classical dogma teaches that enzymes lower the free energy barrier to reaction. We will investigate if this is necessarily so, if there are other ways to catalyze chemical reactions, and how protein dynamics can couple to chemical reaction. The work proceeds through quantum theories of chemical reaction in condensed phase to studies of how the symmetry of couple vibrational modes differentially affects reaction dynamics. Specific examples will include a variety of condensed phase chemical reactions (liquid and crystalline) and a variety of enzymatically catalyzed reactions including the reactions of alcohol dehydrogenase, lactate dehydrogenase, and purine nucleoside phosphorylase.
Biological systems were optimized by evolution to reach a maximum overall efficiency. However, the available structural, spectroscopical, and biochemical information do not allow one to determine what are the most important catalytic mechanisms. A significant part of this difficulty is associated with the ill-defined nature of some proposals and with the slow realization that computer simulation approaches provide perhaps the best way for defining and examining the issues in a unique way (1-3). This talk focuses on the proposal that dynamical effects play a major role in enzyme catalysis (e.g., see references in 4). The analysis of this proposal starts by defining it by unique terms that can be actually verified. It is also to point out that all reactions involve atomic motions but that in order to have a catalytic advantage to such motions they must behave in a different way in enzymes and solutions. A wide range of simulation techniques are used to examine the magnitude of the dynamical effects. It is found that these effects do not contribute to catalysis, regardless of the definition used (4-7). In particular, it is demonstrated that the "solvent contribution to catalysis involves similar dynamics in the enzyme and in solution and that the so-called nonequilibrium solvation effects are not dynamical effects but well defined free energy contributions. Finally, it is illustrated that enzymes work by using their preorganized polar environment to stabilize the transition state of the reacting substrates. This means that enzyme catalysis is due to enzyme-enzyme interaction and not to enzyme-substrate interaction.
Photolyase is a classic photoenzyme and uses blue-light energy to repair cyclobutane pyrimidine dimer (CPD), usually thymine dimer, in ultraviolet (UV)-induced DNA lesion in all three kingdoms. Extensive biochemical and biophysical studies proposed a radical mechanism with a unique catalytic photocycle. However, the key step in this hypothesis has never been observed and the intermediates have never been captured. Integrating femtosecond spectroscopy and molecular biology methods, we mapped out the entire functional evolution in real time by following the dynamics of various species. The repair process of DNA lesion was directly observed in less than one nanosecond. Active-site solvation in the enzyme was observed to continuously couple with the enzymatic reaction in the entire catalytic photocycle. This is probably the first and also simplest light-driven biological machinery which has been completely characterized to show how nature efficiently converts solar energy to perform important biological functions, for the system reported here, to repair UV-induced DNA damage.