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 Flavobacterium 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 degrees C and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-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.
Jung Ahn
Min
M
Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University, Sarimdong, Changwon City, Kyungnam 641-773, South Korea.
Young Moon
Ja
J
Ook Kang
Dae
D
Cho
Yong Kweon
YK
D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005417:Flavobacterium / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D008956:Models, Chemical; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D004791:Enzyme Inhibitors; D008670:Metals; C011669:ascorbate-2-phosphate; D010770:Phosphotransferases; C492726:ascorbic acid 2-kinase, Flavobacterium devorans; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid