tailieunhanh - Báo cáo khoa học: On the mechanism of a-amylase Acarbose and cyclodextrin inhibition of barley amylase isozymes

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barleya-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-malto-dextrin 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 /Kmdecreased 10 3 -fold using rDP18-maltodextrin and 10 5 to 10 6 -fold using maltohep-taose as substrate. . | Eur. J. Biochem. 270 3871-3879 2003 FEBS 2003 doi On the mechanism of a-amylase Acarbose and cyclodextrin inhibition of barley amylase isozymes Naima Oudjeriouat1 Yann Moreau2 Marius Santimone1 Birte Svensson3 Guy Marchis-Mouren1 and Veronique Desseaux1 1IMRN Institut Mediterraneen de Recherche en Nutrition Faculte des Sciences et Techniques de St Jerome Université d Aix-Marseille France 2IRD Institut de Recherche pour le Developpement UR081 Gamet c o CEMAGREF Montpellier France 3Carlsberg Laboratory Department of Chemistry Copenhagen Valby Denmark Two inhibitors acarbose and cyclodextrins CD were used to investigate the active site structure and function of barley a-amylase isozymes AMY1 and AMY2. The hydrolysis of DP 4900-amylose reduced r DP18-malto-dextrin 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 105 to 106-fold using maltohep-taose 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. a-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose but was a very weak inhibitor compared to acarbose. b- and y-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 .