Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold-adapted deep-sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ∼38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady-state rate constant (k(cat)) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K(M)) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k(cat) was therefore offset by a similar change in K(M). Both the activation enthalpy and entropy of the MpDHFR-catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady-state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k(cat) and K(M).

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

In recent years, the green chemistry based-approach for the synthesis of nanoparticles has shown tremendous promise as an alternative to the costly and environmentally unfriendly chemically synthesized nanoparticles. In this study, copper oxide nanoparticles (CuO NPs) were synthesized through a green approach using the water extract of papaya (Carica papaya L.) peel biowaste as reducing as well as stabilizing agents, and copper (II) nitrate trihydrate salt as a precursor. The structural properties, crystallinity, purity, morphology, and the chemical composition of as-synthesized CuO NPs were analyzed using different analytical methods. The analytical results revealed that the synthesized CuO was observed as spherical-like in particles with measured sizes ranging from 85–140 nm and has monoclinic crystalline phase with good purity. The Fourier transform infrared (FTIR) spectroscopic results confirmed the formation of the Cu-O bond through the involvement of the potential functional groups of biomolecules in papaya peel extract. Regarding photocatalytic activity, the green-synthesized CuO NPs were employed as a photocatalyst for the degradation of palm oil mill effluent (POME) beneath the ultraviolet (UV) light and results showed 66% degradation of the POME was achieved after 3 h exposure to UV irradiation. The phytotoxicity experiment using mung bean (Vigna radiata L.) seed also showed a reduction of toxicity after photodegradation.