5-Keto-d-fructose: VI. A SPECIFIC REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE-LINKED REDUCTASE FROM YEAST

Abstract

Abstract A specific reduced nicotinamide dinucleotide phosphate-linked 5-keto-d-fructose reductase has been isolated and purified approximately 300-fold from extracts of bakers' yeast. The reaction catalyzed by the enzyme was found to be 5-Keto-d-fructose + NADPH + H+ → l-sorbose + NADP+ Km values for NADPH and 5-keto-d-fructose were 4.6 x 10-5 m and 1 x 10-3 m, respectively. All attempts to demonstrate reversibility of the reaction were unsuccessful. Although l-sorbose, at exceedingly high concentrations, does not affect the rate of NADPH oxidation by 5-keto-d-fructose, NADP+ was found to be a competitive inhibitor with a Ki value of 1.3 x 10-4 m. Of all other substrates examined, only 5-keto-d-fructose phosphate was active. The Km value for the phosphate ester was found to be 2.9 x 10-4 m with a maximal rate of reduction of only 1.8% of that observed for 5-keto-d-fructose. It has been established that the yeast reductase as well as sheep liver NAD-specific sorbitol dehydrogenase and Candida utilis NADP-specific polyol dehydrogenase catalyze a stereospecific direct transfer of hydrogen from the A side of the reduced pyridine ring to the ketohexose substrates.