The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.
In order to perform Mossbauer studies, Zn(I1) in the Fe(1II)-Zn(1I) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-s7Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H,O, or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with s7Fe(II) in the presence of 0, leads to the "Fe(II1)- . They were reported to include essentially manganese. Recent studies suggest, however, that at least the sweet potato phosphatase is an iron enzyme [lo], thus, in regard to the active center, it might correspond to the mammalian enzymes.
A joint theoretical-experimental study focusing on the description of the ferromagnetic resonance response of thin films in the presence of periodic perturbations introduced on the upper film surface is presented. From the viewpoint of theory, these perturbations may exist in the form of any kind of one-or two-dimensional rectangular defect arrays patterned onto one surface of the magnetic film. Indeed, the defects may be pits or bumps, or ionimplanted regions with a lower saturation magnetization. The complete set of response functions, given by the components of the frequency and wavevector dependent dynamic magnetic susceptibility tensor of the film exposed to microwave excitation, are provided and are used to explain the experimental data. This allows us to obtain the response of the system due to microwave absorption, from which the zero wave-vector spin-wave modes in the fieldfrequency spectra, including their intensity, are calculated. Explicit calculations for periodic defects featuring the shape of stripes, dots and rectangles are given
We report on pairs of converging-diverging spin vortices in Co/Rh/NiFe trilayer disks. The lateral magnetization distribution of these effective spin merons is directly imaged by means of element-selective x-ray microscopy. By this method, both the divergence and circulation states of the individual layers are identified to be antisymmetric. Reversal measurements on corresponding continuous films reveal that biquadratic interlayer exchange coupling is the cause for the effective meron pair formation. Moreover, their three-dimensional magnetization structure is determined by micromagnetic simulations. Interestingly, the magnetic induction aligns along a flux-closing torus. This toroidal topology enforces a symmetry break, which links the core polarities to the divergence configuration.
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