A new erythrose 4-phosphate dehydrogenase coupled assay for transketolase.
The standard assay for transketolase (E.C 2.2.1.1) has depended upon the use of D-xylulose 5-phosphate as the ketose donor substrate since the production of D-glyceraldehyde 3-phosphate can be readily coupled to a reaction that consumes NADH allowing the reaction to be followed spectrophotometrically. Unfortunately, commercial supplies of D-xylulose 5-phosphate recently became unavailable. In this article we describe the coupling of a transketolase reaction (using Leishmania mexicana transketolase) that converts D-fructose 6-phosphate to D-erythrose 4-phosphate. D-Erythrose 4-phosphate can then be converted to 4-phosphate D-erythronate using erythrose-4-phosphate dehydrogenase (E.C 1.2.1.72), a reaction that reduces NAD+ to NADH and can be easily followed spectrophotometrically. D-Ribose 5-phosphate and D-glyceraldehyde 3-phosphate can both be used as ketol acceptor substrates in the reaction although D-ribose 5-phosphate is also a substrate for the coupling enzyme.
Naula
Christina
C
University of Glasgow, Institute of Biomedical and Life Sciences, Division of Infection & Immunity, The Glasgow Biomedical Research Centre, Glasgow G12 8TA, UK.
Alibu
Vincent P
VP
Brock
Janice M
JM
Veitch
Nicola J
NJ
Burchmore
Richard J S
RJ
Barrett
Michael P
MP
D000445:Aldehyde Oxidoreductases / Q000032:analysis* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism*; D015394:Molecular Structure; D014174:Transketolase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D000445:Aldehyde Oxidoreductases; C066236:erythrose 4-phosphate dehydrogenase, E coli; D014174:Transketolase