Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism.
BACKGROUND
The nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidative decarboxylase, 6-phosphogluconate dehydrogenase, is a major source of reduced coenzyme for synthesis. Enzymes later in the pentose phosphate pathway convert the reaction product, ribulose 5-phosphate, to ribose 5-phosphate. Crystallographic study of complexes with coenzyme and substrate explain the NADP dependence which determines the enzyme's metabolic role and support the proposed general base-general acid mechanism.


RESULTS
The refined structures of binary coenzyme/analogue complexes show that Arg33 is ordered by binding the 2'-phosphate, and provides one face of the adenine site. The nicotinamide, while less tightly bound, is more extended when reduced than when oxidized. All substrate binding residues are conserved; the 3-hydroxyl of 6-phosphogluconate is hydrogen bonded to N zeta of Lys183 and the 3-hydrogen points towards the oxidized nicotinamide. The 6-phosphate replaces a tightly bound sulphate in the apo-enzyme.


CONCLUSIONS
NADP specificity is achieved primarily by Arg33 which binds the 2'-phosphate but, in its absence, obscures the adenine pocket. The bound oxidized nicotinamide is syn; hydride transfer from bound substrate to the nicotinamide si- face is achieved with a small movement of the nicotinamide nucleotide. Lys183 may act as general base. A water bound to Gly130 in the coenzyme domain is the most likely acid required in decarboxylation. The dihydronicotinamide ring of NADPH competes for ligands with the 1-carboxyl of 6-phosphogluconate.
Adams
M J
MJ
University of Oxford, Laboratory of Molecular Biophysics, UK.
Ellis
G H
GH
Gover
S
S
Naylor
C E
CE
Phillips
C
C
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003653:Decarboxylation; D005942:Gluconates / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007521:Isocitrate Dehydrogenase / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008968:Molecular Conformation; D009243:NAD / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D009249:NADP / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D012756:Sheep; D013379:Substrate Specificity
D005942:Gluconates; D009243:NAD; C039346:nicotinamide-8-bromoadenine dinucleotide; D009249:NADP; D001120:Arginine; D007521:Isocitrate Dehydrogenase; D010734:Phosphogluconate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; C008884:6-phosphogluconic acid