Computational studies of enzyme mechanism: linking theory with experiment in the analysis of enzymic H-tunnelling

Hydrogen transfer--an essential component of most biological reactions--is a quantum problem. A crucial question of great current interest is how enzymes modulate the quantum dynamics of hydrogen transfer to achieve their outstanding catalytic properties. That tunnelling occurs is now widely accepted, with the conceptual frameworks incorporating protein motion into the enzymic H-tunnelling process. Computational simulation can be used to help elucidate how enzymes work and facilitate H-tunnelling at the atomic level. We review the strength of a multidisciplinary approach--combining computational simulations with enzyme kinetics and structural biology--in revealing tunnelling mechanisms in enzymes. We focus on two paradigm systems--aromatic amine dehydrogenase, in which H-tunnelling is facilitated by fast (sub-picosecond) short range motions, and dihydrofolate reductase, in which a network of long-range coupled motions drives the tunnelling event.     

Dioxygenase-catalysed oxidation of disubstituted benzene substrates: benzylic monohydroxylation versus aryl cis-dihydroxylation and the meta effect

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric substituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.     

Anion binding inhibition of the formation of a helical organogel

A chiral tris(urea) organogelator gels dmso-water and methanol-water mixtures at low weight percent. The formation of the helical gel fibres is partially inhibited by addition of chloride, which is bound by the gelator, resulting in fully crystalline material characterised by X-ray crystallography.     

Frequency Domain Fluorescence Lifetime Study of Crude Petroleum Oils

Frequency domain (FD) fluorescence lifetime data was collected for a series of 20 crude petroleum oils using a 405 nm excitation source and over a spectral range of ~426 to ~650 nm. Average fluorescence lifetimes were calculated using three different models: discrete multi-exponential, Gaussian distribution, and Lorentzian distribution. Fitting the data to extract accurate average lifetimes using the various models proved easier and less time consuming for the FD data than with Time Correlated Single Photon Counting (TCSPC) methods however the analysis of confidence intervals to the computed average lifetimes proved cumbersome for both methods. The uncertainty in the average lifetime was generally larger for the discrete lifetime multi-exponential model when compared to the distribution-based models. For the lifetime distributions, the data from the light crude oils with long lifetimes generally fit to a single decay term. Heavier oils with shorter lifetimes required multiple decay terms. The actual value for the average lifetime is more dependant on the specific fitting model employed than the data acquisition method used. Correlations between average fluorescence lifetimes and physical and chemical parameters of the crude oils were made with a view to developing a quantitative model for predicting the gross chemical composition of crude oils. It was found that there was no significant benefit gained by using FD over TCSPC other than more rapid data analysis in the FD case. For the FD data the Gaussian distribution model for fluorescence lifetime gave the best correlations with chemical composition allowing a qualitative correlation to some bulk oil parameters.

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping‐Electron Paramagnetic Resonance Approach

Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins (homo and hetero metal ions).     

Anion‐Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin‐12, is used to create enzymatically crosslinked, macro‐scale hydrogels. Upon exposure to specific salt conditions, suckerin‐12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin‐12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin‐12 hydrogels. The potential for suckerins to add new properties to materials based on naturally‐derived proteins is highlighted.     

Samarium(II)-mediated linker cleavage-cyclization in fluorous synthesis: reactions of samarium enolates

SmI2 has been used to cleave a sulfur linker and trigger cyclizations in strategies for the traceless fluorous synthesis of N-heterocycles. The studies give further insights into the reactivity of samarium enolates.