6--Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). It not only gives NADPH and RU5P, but also depletes 6PG, whose accumulation induces cell senescence. It is a proposed drug target for African trypanosomiasis caused by T. brucei and for other microbial infections and cancer. We report here that the association of dimers to tetramers is an equilibrium present in the free enzyme, independently of the ligands. It has been shown by glutaraldehyde cross-linking, dynamic light scattering (DLS) and density gradient sedimentation. Both DLS and sedimentation indicate the enzyme size increases by increasing the enzyme concentration. In addition, gel filtration, density gradient sedimentation and isothermal titration calorimetry (ITC) reveal that the oligomerization rates are differently influenced by ligands. Indeed dynamic experiments where different oligomeric forms can be separated, show a strong NADPH shift of the enzyme versus the tetrameric form while NADP does not affect dimeric state of 6PGDH. Accordingly heat capacity change measured by ITC is different between NADP and NADPH binding (-92.56 against -520.35 cal/mol∙K) in agreement with a decreased solvent exposed surface area in tetramer. Tetramer is about 3-fold more active than dimer, indeed NADPH, the 6PGDH product and inhibitor, decreases interconversion rate while 6PG antagonizes NADPH effect. This is a further substrate way of promoting increased catalytic efficiency. The sheep liver 6PGDH, instead, by sedimentation studies appears always a dimer, the dimertetramer shift hence representing further drug exploitable potential.

NADPH reduces oligomerization rate of a pre-existing dimer-tetramer equilibrium inTrypanosoma brucei 6-phosphogluconate dehydrogenase

HANAU, Stefania;DALLOCCHIO, Franco Pasquale Filippo
2013

Abstract

6--Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). It not only gives NADPH and RU5P, but also depletes 6PG, whose accumulation induces cell senescence. It is a proposed drug target for African trypanosomiasis caused by T. brucei and for other microbial infections and cancer. We report here that the association of dimers to tetramers is an equilibrium present in the free enzyme, independently of the ligands. It has been shown by glutaraldehyde cross-linking, dynamic light scattering (DLS) and density gradient sedimentation. Both DLS and sedimentation indicate the enzyme size increases by increasing the enzyme concentration. In addition, gel filtration, density gradient sedimentation and isothermal titration calorimetry (ITC) reveal that the oligomerization rates are differently influenced by ligands. Indeed dynamic experiments where different oligomeric forms can be separated, show a strong NADPH shift of the enzyme versus the tetrameric form while NADP does not affect dimeric state of 6PGDH. Accordingly heat capacity change measured by ITC is different between NADP and NADPH binding (-92.56 against -520.35 cal/mol∙K) in agreement with a decreased solvent exposed surface area in tetramer. Tetramer is about 3-fold more active than dimer, indeed NADPH, the 6PGDH product and inhibitor, decreases interconversion rate while 6PG antagonizes NADPH effect. This is a further substrate way of promoting increased catalytic efficiency. The sheep liver 6PGDH, instead, by sedimentation studies appears always a dimer, the dimertetramer shift hence representing further drug exploitable potential.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/1870750
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact