Purification and characterization of ascorbic acid 2-kinase from Flavobacterium devorans ATCC 10829

Maximum activity for phosphorylating C-2-OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterhan devorans ATCC 10829. The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography. Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa. Among available second substrates, pyrophosphate showed the highest activity. Optimum temperature and pH were 45 degreesC and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site. pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base. (C) 2003 Elsevier SAS. All rights reserved.