Eigil Praestgaard

Comparison of depolarized rayleigh-wing scattering and far-infrared absorption in molecular liquids

Abstract The absorbed energy in a depolarized Rayleigh-wing scattering experiment is constructed from the measured intensity and compared with the far-infrared

Hydrogen bonding in liquid formamide. A low frequency Raman study

Low frequency Raman spectra from 20 to 400 cm−1 of liquid HCONH2, HCOND2, and HCO 15NH2 were studied. Experimental data were directly transformed to the R(v) representation, previously described. Assignments of low frequency bands are discussed based on a comparison between frequencies for the isotopic species and measurements of depolarization ratios. Chain structures are the dominant species. An out‐of‐plane intermolecular mode involving atoms in the hydrogen bond was assigned. The frequency for this mode decreased with increasing temperature.

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Background: The molecular understanding of factors that limit enzymatic hydrolysis of cellulose remains incomplete. Results: Pre-steady-state analysis of cellulolytic activity provides rate constants for basic steps of the overall reaction. Conclusion: Slow dissociation of inactive enzyme-cellulose complexes governs the hydrolytic rate at pseudo-steady state. Significance: Kinetic constants elucidate molecular mechanisms and structure-function relationships for cellulases. The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

A kinetic model for the burst phase of processive cellulases

Cellobiohydrolases (exocellulases) hydrolyze cellulose processively, i.e. by sequential cleaving of soluble sugars from one end of a cellulose strand. Their activity generally shows an initial burst, followed by a pronounced slowdown, even when substrate is abundant and product accumulation is negligible. Here, we propose an explicit kinetic model for this behavior, which uses classical burst phase theory as the starting point. The model is tested against calorimetric measurements of the activity of the cellobiohydrolase Cel7A from Trichoderma reesei on amorphous cellulose. A simple version of the model, which can be solved analytically, shows that the burst and slowdown can be explained by the relative rates of the sequential reactions in the hydrolysis process and the occurrence of obstacles for the processive movement along the cellulose strand. More specifically, the maximum enzyme activity reflects a balance between a rapid processive movement, on the one hand, and a slow release of enzyme which is stalled by obstacles, on the other. This model only partially accounts for the experimental data, and we therefore also test a modified version that takes into account random enzyme inactivation. This approach generally accounts well for the initial time course (approximately 1 h) of the hydrolysis. We suggest that the models will be useful in attempts to rationalize the initial kinetics of processive cellulases, and demonstrate their application to some open questions, including the effect of repeated enzyme dosages and the ‘double exponential decay’ in the rate of cellulolysis.

Molecular dynamics calculation of the liquid structure up to a solid surface

The technique of molecular dynamics is used to calculate the properties of a liquid phase in contact with a fcc crystal phase. A system of 1680 particles which interact through a Lennard‐Jones potential is divided into two closed subsystems by imposing artificial boundaries across which the particles can interact, but not pass. One of the subsystems is melted and a state corresponding to coexisting liquid–solid bulk states is obtained. The constraint is removed and it is observed that the state is maintained, which suggests that the system is in thermodynamic equilibrium. The interface shows a gradual decline of order, and the structure in the middle of the fluid system cannot be distinguished from that of a pure liquid with the same temperature and density. The density profile is also calculated for a fluid up to a solid surface without lattice structure where the layering in the interface is seen to be more pronounced.

Comments on the R(ν̄) spectral representation of the low frequency Raman spectrum

A few misunderstandings concerning our use of the R (?) spectral representation are clarified. Two different spectral representations for the low frequency Raman spectrum are compared.

Perturbation Theory for Fluids

Recently, Barker and Henderson have introduced a semimacroscopic approximation to the second order in the expansion of the configuration integral by considering the pair potential as the sum of a strong (repulsive) part and a weaker (long‐range) part. We analyze this approximation and show that the essential part of it is to reduce the higher‐order distribution functions to a second‐order nonuniform distribution function, the nonuniformity coming from fixing a particle at the origin. The approximation can be done to all orders, and the series can be summed to give an expression for the free energy and pressure. The expression involves the grand partition function for the nonuniform system taken at the chemical potential for the reference system minus the perturbing potential. The functions of the nonuniform system are approximated by taking the functional form they have at uniformity and by using the product of the density and the radial distribution function of the reference system for the nonuniform density. The summed series can then be computed. For the three‐dimensional square‐well fluid it is confirmed that the convergence is very rapid down to reduced temperatures about 0.5. Comparison with the one‐dimensional square‐well fluid shows that below this temperature the convergence is slow and it is necessary to use the whole series.

Oscillating chemical reactions and phase separation simulated by molecular dynamics

Molecular dynamics (MD) of stationary chemical kinetics is used to simulate oscillating chemical reactions in a system of N classical mechanical particles with Lotka–Volterra kinetics. The MD includes oscillations in a (closed) system with conserved energy and time reversible dynamics as well as oscillating chemical reactions in an open and driven non-equilibrium system, and with and without a competing phase separation of the different components in the reactions. The approach allows a detailed investigation of the kinetics and demonstrates on a molecular level, the phenomenon oscillating reactions for various chemical and reaction kinetics details. When phase separation takes place during the oscillations the kinetics is no longer simple diffusion driven.

Interaction between dimethylsulfoxide and formamide in the liquid state

Low frequency Raman spectra (20–400 cm−1) are presented of formamide dissolved in dimethylsulfoxide (DMSO). The concentration of formamide ranges from 2%–75% (v/v). The two formamide bands at 110 and 190 cm−1 in the neat liquid exhibit only smaller frequency shifts upon dilution. The intensities of both bands are proportional to the concentration of formamide. The conclusion is, that the linear chain structure, found in neat liquid formamide, is conserved in the DMSO solution. Raman spectra of the NH‐stretching region (2950–3500 cm−1) confirm this hypothesis.

Aspects of low frequency vibrations (20–350 cm−1) from Watson-Crick base pairing in an aqueous solution of tRNA

Summary The low frequency Raman spectrum from 20 cm −1 to 350 cm −1 of tRNA from Escherichia coli in an aqueous solution is studied. A band at ca . 115 cm −1 is assigned to displacements of atoms in hydrogen bonds between Watson-Crick base pairs. Temperature studies between room temperature and ca. 90°C are performed showing near reversibility of the base pairing upon temperature. The couplings between low frequency vibrations of tRNA and formamide, N-methylformamide and N,N-dimethylformamide are discussed in terms of physiological effects.