title: Beta-thiomaltosides as active site probes for alpha-amylase.
abstract: A series of substituted 1-thio-beta-D-maltopyranosides was synthesized and confirmed by elemental analysis, optical rotation, NMR, and liquid chromatography. These compounds were shown by several biochemical techniques to bind to the active site of alpha-amylase. Steady-state kinetic studies showed the compounds to be competitive inhibitors, with affinities lying within the range of the natural ligands, maltose and maltotriose. Affinity chromatography employing p-aminophenyl-1-thio-beta-D-maltopyranoside linked to Sepharose provides a relatively simple procedure for alpha-amylase purification. The binding of p-bromphenyl-1-thio-beta-D-maltoside was observed in crystals of alpha-amylase using X-ray crystallography, and through the use of difference Fourier analysis its interaction at 5.0-A resolution with the active site of the enzyme has been visualized. The inhibitor binds in a long, deep cleft that divides the two major domains of the enzyme. These studies are believed to provide a first step toward the rational design of ligands for the physiological regulation of starch breakdown and utilization through modulation of alpha-amylase activity.
mesh_terms: D000818:Animals; D001665:Binding Sites; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D013552:Swine; D013863:Thioglucosides / Q000138:chemical synthesis* / Q000494:pharmacology; D013865:Thioglycosides / Q000138:chemical synthesis*; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007202:Indicators and Reagents; D013863:Thioglucosides; D013865:Thioglycosides; D000516:alpha-Amylases