Binding of Coenzyme and Substrate and Coenzyme Analogues to 6‐Phosphogluconate Dehydrogenase from Sheep Liver

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Abstract
The analogs of the coenzyme NADP+, nicotinamide-8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine-adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was active in the H transfer and io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase [EC 1.1.1.44]. The binding of NADP+, NADPH and NADPH and of both analogs to crystals of the enzyme 6-phosphogluconate dehydrogenase was investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site was determined from the io3PdADP+-NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. The analog is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and was used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.