Fluoro-glycosyl acridinones are ultra-sensitive active site titrating agents for retaining β-glycosidases.
نویسندگان
چکیده
Novel fluorogenic 2-deoxy-2-fluoroglycosyl acridinone active site titrating reagents were synthesised and kinetic parameters determined for their inactivation of two retaining β-glucosidases, a β-galactosidase, a β-xylosidase and several cellulases. Fluorescence-monitored active site titration using this class of reagents reliably measured active enzyme concentrations down to 3 nM.
منابع مشابه
2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors.
Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake. This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate. This is achieved by use of 2-deoxy-2-fluoro-D-gly...
متن کاملSemi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase.
A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a h...
متن کاملUsing the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases
The active site residues in GH1 β-glycosidases are compartmentalized into 3 functional regions, involved in catalysis or binding of glycone and aglycone motifs from substrate. However, it still remains unclear how residues outside the active site modulate the enzymatic activity. To tackle this question, we solved the crystal structure of the GH1 β-glycosidase from Spodoptera frugiperda (Sfβgly)...
متن کاملPurification and properties of a beta-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae.
Two beta-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl beta-D-glucoside (NPbetaGlu) (79% of the total activity in...
متن کاملLong-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile.
The mutant E134A 1,3-1,4-beta-glucanase from Bacillus licheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner. A long-lived alpha-glycosyl formate is detected and identified by (1)H-NMR and matrix-assisted laser desorption ionization-time-of-flight-MS. The int...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Chemical communications
دوره 50 66 شماره
صفحات -
تاریخ انتشار 2014