title: On the mechanism of alpha-amylase.
abstract: Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family.
mesh_terms: D020909:Acarbose / Q000378:metabolism / Q000494:pharmacology*; D000688:Amylose / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003505:Cyclodextrins / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D005936:Glucans; D007527:Isoenzymes; D011134:Polysaccharides; C031146:maltoheptaose; C008315:maltodextrin; D000688:Amylose; D000516:alpha-Amylases; D020909:Acarbose