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

Abstract

The analogues of the coenzyme NADP', nicotinamide-8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine -adenine dinucleotide phosphate (io3PdADP'), were prepared. Nbr'ADP' was found to be active in the hydrogen transfer and io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP', NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been 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 has been 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. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been 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. 6-Phosphogluconate dehydrogenase is an oxidative decarboxylase of the pentose phosphate pathway which catalyses the conversion of 6-phosphogluconate to ribulose 5-phosphate using the coenzyme NADP.The enzyme is dimeric with a monomer molecular weight of 50000. The two subunits act independently [l]. The mechanism of the oxidation has been studied under various conditions with different results. A random-order mechanism has been suggested in buffers such as Tris-acetate [2] while an ordered mechanism, binding of coenzyme followed by substrate, has been indicated in phosphate buffer at pH 7 [3]. Inhibition by various nucleotide phosphates, coenzyme fragments and other metabolic intermediates has been studied [4]. The coenzyme NAD' does not bind to the Abbreviations. ADP-Rib, adenosine diphosphoribose; io3PdAD(P)+, 3-iodopyridine -adenine dinucleotide (phosphate); n3PdAD(P)+, 3-aminopyridine ~ adenine dinucleotide (phosphate) ; Nbr*AD(P)+, nicotinamide -8-bromo-adenine dinucleotide (phosphate); NMR, nuclear magnetic resonance.Eniyme. 6-Phosphogluconate dehydrogenase (EC 1 .I .I .44).enzyme in solution [3]. A number of chemical modifications for this enzyme from several other sources have been described [5]. The sheep liver enzyme crystallizes in space group C 2221 (a = 7.27nm, b = 14.82nm, c = 10.29nm) with a single subunit in the asymmetric unit [6]. The 0.6-nm resolution structure of the enzyme has been determined using two heavy atom derivatives : potassium dicyanoaurate (I), KAU(CN)~, and potassium tetracyanoplatinate (11), KZPt(CN)4 [7]. The two subunits are related by a crystallographic twofold axis parallel to b. The subunit contains two a-helices more than 5....

The NADPH binding site on beef liver catalase.

“…ref. 9 for their definition; in addition, the dihedral angle O' relates to the C2'(a)-02'(a) bond giving the position of the 02' bound phosphate] of the coenzyme are shown in Table 3, where they are compared with the well-refined extended NADP structure found in Lactobqcillus casei dihydrofolate reductase (DHFR) (19) as well as with the tentative, low-resolution, structure of NADP in 6-phosphogluconate dehydrogenase (24). The adenosine nucleoside structures of NADP bound to BLC and to DHFR are very similar, with the 02' phosphate positioned in the sterically favored trans orientation relative to the ribose ring.…”

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca2+ Release

“…Conditions for the synthesis of 8-bromo-NAD 47,48 and 8-bromo-AMP 49 have been previously reported, and NADP was similarly converted to 24 . 50 …”

Activity Determination of 3‐Iodopyridineadenine Dinucleotide and Its Phosphate as Hydride Acceptors in the Presence, of Dehydrogenases Using a Coupled Redox System

“…Io3PdAD+ was a competitive inhibitor with respect to NAD' with glyceraldehyde-3-phosphate dehydrogenase and io3PdADP+ a competitive inhibitor with respect to NADP' with 6-phosphogluconate dehydrogenase. The binding of io'PdADP' to the last enzyme was determined at a 0.6-nm resolution and the lack of activity with this analogue was attributed to the fact that it is the carbon C-5 of the 3-iodo-pyridinium ring that points toward the substrate in the ternary complex rather than the carbon C-4 [2]. In lactate dehydrogenase, the coenzyme amide group binds to the hydroxyl group of Ser-16 [13,14].…”

“…Io3PdAD(P)+ [l, 21, analogues of the coenzymes NAD(P)+, have been used as heavy atom labels with horse liver alcohol dehydrogenase [3] and 6-phosphogluconate dehydrogenase [2]. With the latter, the orientation of the bound coenzyme was determined [2].…”

“…With the latter, the orientation of the bound coenzyme was determined [2]. In the ternary complex alcohol dehydrogenaseio3PdAD+ -imidazole, the analogue was bound in a different way from that of the natural coenzyme: the pyridinium ring is located completely outside the active site [3].…”