Chemistry Of Transhydrogenase

Understanding the link between the structure and mechanism of transhydrogenase is crucial for developing our knowledge on how NADPH is formed. Altered levels of transhydrogenase and changes in its structure have adverse effects within the cell.

Introduction:-

Transhydrogenase is an enzyme found in a diverse range of species such as bacteria and mitochondria.1 In mitochondria found within adrenocortical cells, a reduction in the level of transhydrogenase causes apoptosis of these cells and hence a decrease in the production of glucocorticoids.2 Therefore, knowledge gained by this enzyme can be used to help understand the causes of familial glucocorticoid deficiency.2, 3

In brain mitochondria, the enzyme causes an increase in the antioxidant level by being involved in the removal of H2O2.4 Thus, by understanding the mechanism and structure of transhydrogenase, we could use this enzyme as a therapeutic target to reduce the amount of toxicity within cells.4

Chemical reaction in transhydrogenase:-

In order to increase antioxidant levels within a given cell, the amount of NADPH must increase.4 As transhydrogenase transfers a hydride ion from NADH to NADP+, the equilibrium is forced to the right and the relative amount of protons in the cell increases.5 Therefore, the increased level of NADPH is a direct result of hydride exchange and forward proton translocation.5,6

NADH + NADP+ + H out+ = NAD+ + NADPH + H in+ Equation 1. Overall reaction in transhydrogenase5

Whilst this hydride transfer depends on the stereo-specificity of these rings1, the protein components that make up transhydrogenase also have specific roles in this exchange process and therefore also affect the production of NADPH7. Once domain I (dI) binds to NADH and domain III (dIII) binds to NADP+, the dihydronicotinamide ring of NADH and the nicotinamide ring of NADP+ must be brought close enough in order for hydride transfer to occur.5 As hydride exchange takes place, a net movement of protons travel from the periplasm to the cytosol.7 Consequently, NADPH is formed inside the cytosol. 7

Binding change mechanism:-

The dIII component can be in either an open or occluded state.10 When NADP+ binds to dIII in its open state, they are bound together with moderate affininty.10 After this binding takes place, the dIII component transforms into an occluded state.10 The binding affinity between NADP+ and occluded dIII is higher than when it was bound to open dIII.10 NADH bound to dI then transfers a hydride ion to NADP+.10 The NADPH formed is bound most strongly to the occluded dIII.10

As all reactants and products are within the cytosol, and the reduction potentials for NADH/NAD+ and NADPH/NADP+ is negligible (around 5 mV) there must be some driving force present in order for proton translocation into the cytosol to exist.6 Due to differences in binding energies of the reactants and products, the reaction proceeds preferentially in the forward direction to produce NADPH.6

NADP+ bound to open dIII < NADP+ bound to occluded dIII << NADPH bound to occluded dIII

Scheme 1. The relative stabilities of reactants/products that are bound to open or occluded states of dIII. NADPH bound to occluded dIII gives the most stable conformation. 10

Transhydrogenase structure in bacteria and mitochondria:-

Whilst transhydrogenase in both prokaryotes and eukaryotes is involved in producing NADPH, the structure of the enzymes` domains differ in both species.11 For example, dII of E. coli bacteria consists of two subunits called ? and ?6. The whole ?2?2 dII unit10 consists of approximately 400 residues which is made up of 13 ? -helices.8 In mitochondria however, all three domains are arranged as dI-dII-dIII to form a single polypeptide chain and dII is not split into ? and ?.6, 11

Not only do differences in the transhydrogenase structure exist between eukaryotic organelles and prokaryotic cells, they also exist between different species of prokaryotes.6 Whilst E. coli is made up of two subunits, Rhodospirillum rubrum contains three: ?1, ?2, and ?.6 In this type of bacteria, ``?1 corresponds to dI, ?2 corresponds to the N-terminal portion of dII, and the ?-subunit corresponds to the remainder of dII and all of dIII." 6

Although differences in transhydrogenase structure exist, similarities are present.12 For example, in both eukaryotic organelles and prokaryotic cells, the dI and dIII components extend from the membrane and into the cytosol (i.e. towards the side of the cytoplasm in bacteria) or into the cytoplasm (i.e. towards the side of the matrix in mitochondria).12 In both these species, dII spreads across the membrane and contains specific channels that allow proton translocation to occur.11, 12

The effect of an altered dII structure:-

A mutation in the transhydrogenase gene in inbred mice with a B6J strain involves the removal of four ? -helices from dII.13 This deletion mutation is a key factor in causing the reduced activity of transhydrogenase in liver and islets.13, 14 Impaired glucose tolerance, diet-induced obesity and higher epigonadal fat mass are associated with this change in domain structure of transhydrogenase.13

Issues and future applications:-

The information obtained from the transhydrogenase structure has been elucidated from within species only.7 Due to the small sizes of the domains, it is difficult to determine the shapes of these components and hence the actual intact structure of this enzyme.7

By carrying out further research, it may be possible to gain more indepth knowledge of the intact structure.13 This will help us understand the effects of abnormal transhydrogenase functionality better and thus bring us one step closer to treating these effects.13

References

1. T. Bhakta, S. J. Whitehead, J. S. Snaith, T. R. Dafforn, J. Wilkie, S. Rajesh, S. A. White and J. B. Jackson, Biochemistry, 2007, 46, 3304-3318.

2. R. Yamaguchi, F. Kato, T. Hasegawa, N. Katsumata, M. Fukami, T. Matsui, K. Nagasaki and T. Ogata, Endocr. J., 2013, 60, 855-859 .

3. E. Meimaridou, J. Kowalczyk, L. Guasti, C.R. Hughes, F. Wagner, P. Frommolt, P. Nürnberg, N.P. Mann, R. Banerjee, H.N. Saka, J.P. Chapple, P.J. King, A.J.L. Clark and L.A. Methere, Nature Genetics, 2012, 44, 740-742.

4. P. Lopert and M. Patel, J. Biol. Chem., 2014, 289, 15611-15620.

5. M. Iwaki, N.P.J. Cotton, P.G. Quirk, P.R. Rich and J.B. Jackson, J. Am. Chem. Soc., 2006, 128, 2621-2629 .

6. G.S. Prasad, M. Wahlberg, V. Sridhar, V. Sundaresan, M. Yamaguchi, Y. Hatefi and C.D. Stout, Biochemistry, 2002, 41, 12745-12754 .

7. V. Sundaresan, J. Chartron, M. Yamaguchi and C.D. Stout, J. Mol. Biol., 2005, 346, 617-629 .

8. T. Johansson, C. Oswald, A. Pedersen, S.Tornroth, M. Okvist, B.G. Karlsson, J. Rydstrom and U. Krengel, J. Mol. Biol., 2005, 352, 299-312 .

9. T. Harma, C. Brondijk, G.I. van Boxel, O.C. Mather, P.G. Quirk, S.A. White and J.B. Jackson , J. Biol. Chem., 2006, 281, 13345-13354.

10. J.B. Jackson, S.A. White, P.G. Quirk and J.D. Venning, Biochemistry, 2002, 41, 4173-4185.

11. O.C. Mather, A. Singh, G.I.van Boxel, S.A. White and J.B Jackson, Biochemistry, 2004, 43, 10952-10964.

12. A. Singh, J.D. Venning, P.G. Quirk, G.I.van Boxel, D.J. Rodrigues, S.A. White and J.B. Jackson, J. Biol. Chem., 2003, 278, 33208-33216.

13. J.T. Heiker, M. Kern, J. Kosacka, G. Flehmig, M.Stumvoll, E. Shang, T. Lohmann, M. Dreßler, P. Kovacs, M. Bluher and N. Kloting, Obesity, 2013, 21, 529-534.

14. K.A. Mourney, N. Wong, M. Kebede, S. Zraika, L. Balmer, J. M. McMahon, B.C. Fam, J. Favaloro, J. Proietto, G. Morahan, S. Andrikopoulos, Diabetologia, 2007, 50, 2476-2485.