The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and H-1 and F-19 NMR spectroscopy. Whereas 3- and 4-fluorophenol react with... Show moreThe activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and H-1 and F-19 NMR spectroscopy. Whereas 3- and 4-fluorophenol react with tyrosinase to give products that undergo a rapid polymerization process, 2-fluorophenol is not reactive and actually acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine (L-dopa). The tyrosinase-mediated polymerization of 3- and 4-fluorophenols has been studied in detail. It proceeds through a phenolic coupling pathway in which the common reactive fluoro-quinone, produced stereospecifically by tyrosinase, eliminates an inorganic fluorine ion. The enzymatic reaction studied as a function of substrate concentration shows a prominent lag that is completely depleted in the presence Of L-dopa. The kinetic parameters of the reactions can be correlated to the electronic and steric effects of the fluorine substituent position. Whereas the fluorine electron withdrawing effect appears to control the binding of the substrates (K-m for 3- and 4-fluorophenols and K-1 for 2-fluorophenol), the k(cat) parameters do not follow the expected trend, indicating that in the transition state some additional steric effect rules the reactivity. Show less
Amsterdam, I.M.C. van; Ubbink, M.; Bosch, M. van den; Rotsaert, F.; Sander-Loehr, J.; Canters, G.W. 2002
The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and... Show moreThe double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed CyS42 assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell. Show less
The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is... Show moreThe peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated. Show less
Recently, it has been shown that when an equation that allows the so-called pulled fronts in the mean-field limit is modeled with a stochastic model with a finite number N of particles per... Show moreRecently, it has been shown that when an equation that allows the so-called pulled fronts in the mean-field limit is modeled with a stochastic model with a finite number N of particles per correlation volume, the convergence to the speed v(*) for N--> infinity is extremely slow-going only as ln(-2)N. Pulled fronts are fronts that propagate into an unstable state, and the asymptotic front speed is equal to the linear spreading speed v(*) of small linear perturbations about the unstable state. In this paper, we study the front propagation in a simple stochastic lattice model. A detailed analysis of the microscopic picture of the front dynamics shows that for the description of the far tip of the front, one has to abandon the idea of a uniformly translating front solution. The lattice and finite particle effects lead to a "stop-and-go" type dynamics at the far tip of the front, while the average front behind it "crosses over" to a uniformly translating solution. In this formulation, the effect of stochasticity on the asymptotic front speed is coded in the probability distribution of the times required for the advancement of the "foremost bin." We derive expressions of these probability distributions by matching the solution of the far tip with the uniformly translating solution behind. This matching includes various correlation effects in a mean-field type approximation. Our results for the probability distributions compare well to the results of stochastic numerical simulations. This approach also allows us to deal with much smaller values of N than it is required to have the ln(-2)N asymptotics to be valid. Furthermore, we show that if one insists on using a uniformly translating solution for the entire front ignoring its breakdown at the far tip, then one can obtain a simple expression for the corrections to the front speed for finite values of N, in which various subdominant contributions have a clear physical interpretation. Show less
The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR)... Show moreThe effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric (g) over left right arrow and (A) over left right arrow tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins, Such a reduction, quantified by the standard deviations sigma(gparallel to) and sigma(A\) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(a\) and sigma(Aparallel to) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N-3(-) and SCN- addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions, (C) 2002 Elsevier Science Inc. All rights reserved. Show less
Keijser, B.J.F.; Wezel, G.P. van; Canters, G.W.; Vijgenboom, E. 2002
Fronts, propagating into an unstable state phi=0, whose asymptotic speed v(as) is equal to the linear spreading speed v* of infinitesimal perturbations about that state (so-called pulled fronts),... Show moreFronts, propagating into an unstable state phi=0, whose asymptotic speed v(as) is equal to the linear spreading speed v* of infinitesimal perturbations about that state (so-called pulled fronts), are very sensitive to changes in the growth rate f(phi) for phi<1. It was recently found that with a small cutoff, f(phi)=0 for phi< epsilon, v(as) converges to v* very slowly from below, as ln(-2) epsilon. Here we show that with such a cutoff and a small enhancement of the growth rate for small phi behind it, one can have v(as)>v*, even in the limit epsilon -->0. The effect is confirmed in a stochastic lattice model simulation where the growth rules for a few particles per site are accordingly modified. Show less
We have surveyed an optical/IR selected sample of nearby E/S0 galaxies with and without nuclear dust structures with the VLA at 3.6 cm to a sensitivity of 100 μ Jy. We can construct a Radio... Show moreWe have surveyed an optical/IR selected sample of nearby E/S0 galaxies with and without nuclear dust structures with the VLA at 3.6 cm to a sensitivity of 100 μ Jy. We can construct a Radio Luminosity Function (RLF) of these galaxies to ~1019 W Hz-1 and find that ~50% of these galaxies have AGNs at this level. The space density of these AGNs equals that of starburst galaxies at this luminosity. Several dust-free galaxies have low luminosity radio cores, and their RLF is not significantly less than that of the dusty galaxies. Show less
Prigent, A.; Grégoire, G.; Chaté, H.; Dauchot, O.; Saarloos, W. van 2002
We show that turbulent "spirals" and "spots" observed in Taylor-Couette and plane Couette flow correspond to a turbulence-intensity modulated finite-wavelength pattern which in every respect fits... Show moreWe show that turbulent "spirals" and "spots" observed in Taylor-Couette and plane Couette flow correspond to a turbulence-intensity modulated finite-wavelength pattern which in every respect fits the phenomenology of coupled noisy Ginzburg-Landau (amplitude) equations with noise. This suggests the existence of a long-wavelength instability of the homogeneous turbulence regime. Show less
Depending on the growth condition, bacterial colonies can exhibit different morphologies. As argued by Ben-Jacob et al. there is biological and modeling evidence that a nonlinear diffusion... Show moreDepending on the growth condition, bacterial colonies can exhibit different morphologies. As argued by Ben-Jacob et al. there is biological and modeling evidence that a nonlinear diffusion coefficient of the type D(b)=D(0)b(k) is a basic mechanism that underlies almost all of the patterns and generates a long-wavelength instability. We study a reaction-diffusion system with a nonlinear diffusion coefficient and find that a unique planar traveling front solution exists whose velocity is uniquely determined by k and D=D(0)/D(n), where D(n) is the diffusion coefficient of the nutrient. Due to the fact that the bacterial diffusion coefficient vanishes when b-->0, in the front solution b vanishes in a singular way. As a result the standard linear stability analysis for fronts cannot be used. We introduce an extension of the stability analysis that can be applied to singular fronts, and use the method to perform a linear stability analysis of the planar bacteriological growth front. We show that a nonlinear diffusion coefficient generates a long-wavelength instability for k>0 and D0 and k--> infinity the dynamics of the growth zone essentially reduces to that of a sharp interface problem that is reminiscent of a so-called one-sided growth problem where the growth velocity is proportional to the gradient of a diffusion field ahead of the interface. The moving boundary approximation that we derive in these limits is quite accurate but surprisingly does not become a proper asymptotic theory in the strict mathematical sense in the limit D-->0, due to lack of full separation of scales on all dynamically relevant length scales. Our linear stability analysis and sharp interface formulation will also be applicable to other examples of interface formation due to nonlinear diffusion, like in porous media or in the problem of vortex motion in superconductors. Show less
