Cleavage of O/S-glucosides by glycoprotein beta-glucosidase
Description
Part I, Catalytic Mechanism of beta-Glucosidase. Sweet almond beta-glucosidase is a highly efficient family 1 exo-glycohydrolase. The activity of this enzyme depends on a deprotonated carboxylate and a protonated carboxylic acid for the optimal activity. In this study we report on the hydrolysis of O/S-glycoside catalyzed by beta-glucosidase. While the Km values for the S- and O-glycosides are similar, the kcat values are about 1,000-times lower for the S-glycosides. Remarkably, the pH-profile for kcat/Km for hydrolysis of p-nitrophenyl thioglucoside (pNPSG) shows the identical dependence on a deprotonated carboxylate and a protonated groups does the pH-profile for hydrolysis of the corresponding O-glycoside. There is no solvent kinetic isotope effect on the enzyme-catalyzed hydrolysis of pNPSG, so is pNPG. These results suggest the reason that the glutamic acid residue must be protonated is not that it is acting as a general acid catalyst, but rather, that the unionized glutamic acid avoids electrostatic repulsion with the incipient thiolate. Linear free energy relationships between the binding and catalytic parameters and the aryl substituents on the phenyl glucosides reveal a striking similarity between the S-and O-glycosides. For both substrates, catalysis (kcat/K m) shows a strong dependence on the pKa of leaving group (betalg = -1) suggesting that in the transition state there is nearly full development of a negative charge on the (thio)phenolic oxygen or sulfur. Substrate binding shows a dependence on both hydrophobicity and the electronic properties of aryls substituents. The Bronsted coefficient for substrate binding is beta ≈ -0.41, suggesting a weakening of glucosidic bond when the E·S complex is formed Part II, Protein Chemistry of beta-Glucosidase. As a glycoprotein, beta-glucosidase from sweet almond still has no crystal structure with around 5% carbohydrate. Endoglycosidase H but not peptide N-glycosidase F, could cleave carbohydrate from beta-glucosidase. This results suggest that the oligosaccharides of beta-glucosidase are attached to an N-terminal asparigine or C-terminal asparigine. With substrates pNP-Glc and pNP-Fuc the catalytic rate by enzyme-treated beta-glucosidase didn't change comparing to the native enzyme. While with substrate pNP-Gal the catalytic rate increased, with substrate cellobiose the catalytic rate decreased. Therefore it is possible that carbohydrate does play a role in the process of enzymatic catalysis beta-Glucosidase is a stable enzyme that only loses 50% activity after incubation with 8 M urea for 1 hour. beta-glucosidase can be unfolded and also inhibited by urea. Modification of beta-glucosidase by DTNB and NTCB did not change the enzymatic hydrolysis activity. Therefore, this interaction would not change the conformation around catalytic center. At least the thiols groups were not required for this enzymatic catalysis