Download 23003955 Chemical Modification of FAD (Flavin Adenine

23003955
Chemical Modification of FAD (Flavin Adenine Dinucleotide)
to Elucidate the Mechanism of 2-Methyl -3-Hydroxypridine-5Carboxylic (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-Nacetylaminomethylene 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, 8OCH3FAD 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 FADanalogs. The ratio of product to substrate was also measured by oxygen electrode methode.
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