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Evidence that beta-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable beta-d-Glycosidase.

ABSTRACT: A survey of Sulfolobus isolates showed all to contain thermostable enzyme activities hydrolyzing various glycosidic compounds. Of those not previously reported, the beta-glucosidase activity of Sulfolobus solfataricus isolate P2 was chosen for further study and found to have the same kinetics of inactivation, apparent molecular weight, and many (though not all) other biochemical properties of the beta-galactosidase also present in this strain. The two activities copurified approximately 850-fold to apparent homogeneity. The enzyme, whose subunit M(r) was estimated to be 60,000 to 65,000 by gel permeation chromatography of the active enzyme and 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured form, hydrolyzed a variety of low-molecular-weight, beta-linked glycosides and could account for most of the corresponding activities found in crude extract. Kinetic analyses indicated that chromogenic beta-d-galactosides and beta-d-glucosides are hydrolyzed at a common active site and that beta-glucosides and beta-fucosides represent the preferred substrates. The liberation of aglycone from aryl beta-d-glucosides was stimulated by alcohols in a manner suggesting specific interaction between alcohol and enzyme.

SUBMITTER: Grogan DW

PROVIDER: S-EPMC183446 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Evidence that beta-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable beta-d-Glycosidase.

Grogan Dennis W DW

Applied and environmental microbiology 19910601 6

A survey of Sulfolobus isolates showed all to contain thermostable enzyme activities hydrolyzing various glycosidic compounds. Of those not previously reported, the beta-glucosidase activity of Sulfolobus solfataricus isolate P2 was chosen for further study and found to have the same kinetics of inactivation, apparent molecular weight, and many (though not all) other biochemical properties of the beta-galactosidase also present in this strain. The two activities copurified approximately 850-fold ...[more]

PMID: 16348503

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Project description:The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.

Dataset Information

Evidence that beta-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable beta-d-Glycosidase.

Publications

Evidence that beta-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable beta-d-Glycosidase.

Similar Datasets

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