23003955 Chemical Modification of FAD (Flavin Adenine Dinucleotide) to Elucidate the Mechanism of 2-Methyl -3-Hydroxypridine-5- Carboxylic (MHPC) Acid Oxygenase (MHPCO) 12th Annual Meeting of Thai Society for Biotechnology NOV/2000 pp1 THA Jeerus Sudcharitkul, Pimchai Chaiyen Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, THAILAND 2-Methyl-3hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is the enzyme involving in Vitamin B6 catabolism in soil bacteria. MHPCO catalyzes oxygenation of aromatic substrate (MHPC) into aliphatic product, α-N- acetylaminomethylene succinic acid. This enzyme has flavin adenine dinucleotide (FAD) as a coenzyme and crucial structure for catalysis. The model proposed to explain the mechanism of hydroxylation is electrophilcity aromatic substitution. If the enzyme truly employs this mechanism, the reactivity of the enzyme will depend on electrophilicity of the flavin ring. The electrophilicity was changed by substituted native FAD with FAD-analogs with the substituent group at 8-position. FAD-analogs with electron donating group, 8- OCH3FAD and 8-NH2FAD, would decrease in electrophilicity and therefore should decrease in reactivity. 8-CN-FAD and 8-CI-FAD, FAD-analogs with electron withdrawing group, would have more electrophilicity and should increase reactivity in the hydroxylation. In this research project, native FAD was removed by acid ammonuim sulfate precipitation. Then apoenzyme was reconstituted with FAD-analogs and characterized for thermodynamic properties such as redox potential, dissociation constant between reconstituted enzymes and substrates and extinction coefficient of enzyme bound FAD- analogs. The ratio of product to substrate was also measured by oxygen electrode methode. Technical Information Services (TIS) / KMUTT .