A novel assay system for the measurement of transketolase activity using xylulokinase from Saccharomyces cerevisiae.
The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 x 10(-4)unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.
Lee
Jin-Young
JY
Institute of Biotechnological Industry, Inha University, Incheon, 402-751, Korea.
Cheong
Dae-Eun
DE
Kim
Geun-Joong
GJ
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D010428:Pentosephosphates / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010957:Plasmids; D012267:Ribosemonophosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014174:Transketolase / Q000032:analysis*; D014996:Xylulose / Q000378:metabolism
D010428:Pentosephosphates; D012267:Ribosemonophosphates; D029701:Saccharomyces cerevisiae Proteins; C031626:ribose-5-phosphate; D014996:Xylulose; C031625:xylulose-5-phosphate; D014174:Transketolase; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase