16] Enzymic synthesis of 2-keto-3-deoxygluconate 6-phosphate using 6-phosphogluconate dehydratase

Abstract

Publisher Summary This chapter describes the enzymic synthesis of 2-keto-3-deoxygluconate 6-phosphate using 6-phosphogluconate dehydratase. 6-Phosphogluconic dehydratase is an ethylenediaminetetraacetic acid (EDTA)-sensitive enzyme, found in the extracts of glucose-grown Pseudomonas putida, which converts 6-phosphogluconate to 2-keto-3-deoxygluconate 6-phosphate (KDPG). The KDPG aldolase-pyruvate ketimine in the extracts of the organism is reductively trapped, quantitatively inactivating the aldolase while leaving adequate dehydrase activity, which then is used to convert relatively inexpensive 6-phosphogluconate to KDPG. KDPG is then isolated as lithium salt with a good degree of purity. Glucose 6-phosphate is assayed spectrophotometrically using nicotinamide adenine dinucleotide (NAD) and glucose-6-phosphate dehydrogenase. 6-Phosphogluconate is assayed using nicotinamide adenine dinucleotide phosphate (NADP) and 6-phosphogluconate dehydrogenase. 6-Phosphogluconate dehydratase activity is determined spectrophotometrically using a system coupled to KDPG adolase, lactic dehydrogenase, and nicotinamide adenine dinucleotide dehydrogenase (NADH). The progress of the reaction is estimated by assaying perchloric acid-treated samples of KDPG. After 120 min, no further product formation is evident. No detectable 6-phosphogluconate, pyruvate, or glyceraldehyde 3-phosphate are demonstrated in the preparation by appropriate enzymic assays.