RESUMO
We report time-domain rotational spectroscopy of the argon dimer, Ar2, by implementing time-resolved Coulomb explosion imaging of rotational wave packets. The rotational wave packets are created in Ar2 with a linearly polarized, nonresonant, ultrashort laser pulse, and their spatiotemporal evolution is fully characterized by measuring angular distribution of the fragmented Ar+ promptly ejected from Ar22+ generated by the more intense probe pulse. The pump-probe measurements have been carried out up to a delay time of 16 ns. The alignment parameters, derived from the observed images, exhibit periodic oscillation lasting for more than 15 ns. The pure rotational spectrum of Ar2 is obtained by Fourier transformation of the time traces of the alignment parameters. The frequency resolution in the spectrum is about 90 MHz, the highest ever achieved for Ar2. The rotational constant and the centrifugal distortion constant are determined with much improved precision than the previous experimental results: B0 = 1.72713 ± 0.00009 GHz and D0 = 0.0310 ± 0.0005 MHz. The present B0 value does not match within the quoted experimental uncertainty with that from the VUV spectroscopy, so far accepted as an experimental reference to assess theories. The present improved constants would stand as new references to calibrate state-of-the-art theoretical investigations and an indispensable experimental source for the construction of an accurate empirical intermolecular potential.
RESUMO
Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.
