The interaction of mammary glucose 6-phosphate dehydrogenase with pyridine nucleotides and 3beta-hydroxyandrost-5-en-17-one.

Glucose g-phosphate dehydrogenase was first described in yeast by Warburg and Christian (l), who showed that nicotinamide adenine dinucleotide phosphate was the specific coenzyme. The enzyme was later prepared from liver and again shown to be specific for the same coenzyme (2). Subsequent preparations (see Cheldelin (3)) from various microbial sources have included two which react with both nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide (4, 5). Recently, glucose 6-phosphate dehydrogenase has been crystallized from yeast (6) and bovine udder (7) and obtained in highly purified form from human erythrocytes (8). The inhibition of glucose-6-P dehydrogenase by certain steroids was reported by McKerns (9) for the adrenal enzyme and by Marks and Banks (10) for the enzyme isolated from human erythrocytes. The latter authors showed that sensitivity to steroid inhibition was exhibited by glucose-6-P dehydrogenase from other mammalian sources, but not from spinach or yeast. A particularly effective inhibitor was 3fi-hydroxyandrost-5-enl7-one (dehydroepiandrosterone). We have recently reported (11) that under suitable conditions NAD could replace NADP in the oxidation of glucose-6-P catalyzed by glucose-6-P dehydrogenase from mammalian sources, but not with the yeast enzyme. Preliminary evidence suggested that the reactions with NAD and NADP were catalyzed by the same protein (11). This paper describes experiments designed to elucidate the relationship between the NAD-linked and NADP-linked activities of glucose-6-P dehydrogenase and the mechanism of steroid inhibition. Evidence is presented that NAD and NADP compete for one site on the enzyme and that, in addition, NAD is bound at a second site. Dehydroepiandrosterone inhibits the NADP-linked reaction without altering the activity with respect to NAD. Under a variety of conditions, the two activities could be affected differentially and these findings have been interpreted as resulting from alterations in the spatial configuration of the enzyme.