Detection of metabolites of the Entner-Doudoroff pathway by HPLC with pulsed amperometry: application to assays for pathway enzymes.
Three major metabolites in the Entner-Doudoroff pathway, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, and pyruvate can be detected and quantified by HPLC with pulsed amperometric detection. Resolution is achieved by ion-exchange chromatography at alkaline pH with isocratic elution in 5 to 10 min. Detection limits are in the subnanomolar range, and detector response is linear over 3-4 orders of magnitude. This method can be employed for the assay of the enzymes of the pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14), eliminating the need for coupling enzymes as in the previously employed spectrophotometric assays. The lag in pyruvate production seen in the coupled enzyme spectrophotometric assay for 6-phosphogluconate dehydratase is absent in the HPLC/pulsed amperometric detection assay. This lag represents an artifact of a slow tautomerism of 2-keto-3-deoxy-6-phosphogluconate which must precede its utilization by the coupling enzyme, 2-keto-3-deoxy-6-phosphogluconate aldolase. Kinetic data on the approach to equilibrium of 2-keto-3-deoxy-6-phosphogluconate aldolase-catalyzed interconversion of 2-keto-3-deoxy-6-phosphogluconate, pyruvate, and glyceral-dehyde-3-phosphate can be also accurately quantified by HPLC with pulsed amperometric detection.
Taha
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Department of Biochemistry, Michigan State University, East Lansing 48824.
Deits
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D000446:Aldehyde-Lyases / Q000032:analysis* / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D005942:Gluconates / Q000032:analysis* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006836:Hydro-Lyases / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011773:Pyruvates / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity
D005942:Gluconates; D011773:Pyruvates; C081217:2-keto-3-deoxy-6-phosphogluconate; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase; D006836:Hydro-Lyases; C022362:phosphogluconate dehydratase; D005947:Glucose; C008884:6-phosphogluconic acid