A new erythrose 4-phosphate dehydrogenase coupled assay for transketolase

Abstract

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.