Reaction Mechanism and Substrate Specificity of the β- Glycosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus
Marco MORACCIa, Giuseppe PERUGINOa, Maria CIARAMELLAa, and Mose ROSSI*a,b
a Istitute of Protein Biochemistry and Enzymology, Via Marconi 10 80127, Napoli, ITALY
b Dipartimento di Chimica Organica e Biologica, Universita'di Napoli, Via Mezzocannone 16, 80100, Napoli, ITALY
The Sulfolobus solfataricus, strain MT4, β-glycosidase (Ssβ-gly) (EC 3.2.1.x) is a thermophilic and thermostable glycohydrolase showing broad specificity against β- [ 1-4]; -[ 1-3] and -[ 1-6] O-glucosides, and remarkable exo-glucosidase activity against oligosaccharides. The amino acid sequence places this protein in glycosyl hydrolase family-1 along with archaeal, bacterial and eukaryal enzymes. The enzyme has been recently crystallized in the native form and the three dimensional structure has been resolved (1). Ssβ-Gly enzyme crystallyzes as a tetramer with a dimer in the asymmetric unit showing the classic TIM barrel fold. The molecular basis of the enzyme exo-glucosidase activity and broad substrate specificity revealed by enzymological studies could now be rationalised in terms of the properties of a long substrate binding tunnel, observed in the crystal structure, which runs on the surface of the enzyme. Ssβ-Gly hydrolyzes β-glycosides maintaining the anomeric configuration of the substrate, and for this reason can be classified as a retaining glycosyl hydrolase. Retaining β-glycosidases utilize a double displacement mechanism, involving a glycosyl-enzyme intermediate, in a process requiring the coordinate assistance of two carboxylic acid residues in the active site, one acting as nucleophile and the other with the double role of general acid/base catalyst. In Ssβ- gly, glutamic acids 206 and 387 are essential for catalysis as demonstrated by site- directed mutagenesis (2). The Glu387Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206Gln mutant showed 10-fold and 60-fold reduced activity on aryl-galacto and glucosides, respectively, if compared to the wild type. Furthermore, the residual activity of the Glu206Gln mutant loosed the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst. In order to test if additional residues could promote the acid/base catalysis in Ssβ-gly, explaining the residual activity of Glu206Gln mutant, the His 150residue, that is invariant in glycohydrolase family 1, was changed to Ala. Surprisingly enough, the mutation determined a 2 to 10-fold kcat reduction on aryl-galactosides, but did not affected, or
