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PDE5, phosphodiesterase 5 (EC 3.1.4.35), is an abundant protein in cell of airway and visceral smooth muscle and vascular cell. It can be found in epithelial cell and in Purkinje cell of the cerebella [1] and platelets and Corpus Cavernosum. In particular, it is implied in the NO pathway of penile erection and so in the Erectile Dysfunction (ED) [22].
There are 11 families of PDE (from 1 to 9), there is 21 genes for PDE which code 60 different PDE. For the PDE5A, the only PDE5 subcategory, there are 4 isoforms but their catalytic domain is the same [24].
The catalytic reaction is the hydrolysis of guanosine cyclic monophosphate into linear guanosine monophosphate. This cGMP-specific enzyme have 3 domains (from N terminal to C terminal) : GAF A, GAF B and a conserved catalytic domain regard to other PDEs of the family. Only cGMP can bind GAF A or GAF B and it stimulates the hydrolysis.
We study here the PDE5A catalytic fragment formed of amino acid residues from the 535th to the 860th [23]. In the inhibition, we talk about the Sildenafil mostly, because it's the most known (active ingredient in the Viagra®).
Structure of catalytic site
The only catalytic fragment is effective, so the regulations sites and the dimerization to a trimeric enzyme are useless for the catalytic activity. Moreover, this catalytic moiety has the same activity that the wild-type enzyme, so maybe the enzyme is monomeric in the cell [24].
Catalytic domain is conserved for the PDE family, between 20% and 40%, and the variant reactions of the PDE inhibitors on the different PDEs may be caused by the more variant regulatory sites [25]./n
The catalytic domain has 3 helical subdomains [24]:/n
- A N-terminal cyclin-fold region with eight helixes [26]: 5 α-helixes (1, 3, 5, 6 and 8) and 3 310-helixes (2,4, and 7), from the 537th to the 678th residues./n
- A linker domain: two antiparallels α9 and α10 helixes, and between a disordered region, from the 679th to the 725th residues.
- A C-terminal buddle pocket with eight helixes: 5 long α-helixes (11, 12, 14, 17 and 18) and 3 smaller helixes (13, 15 and 16), from the 726th to the 860th residues.
+ α5, 6 and 8 surround α3 and form an interface with the linker domain and the CTD.
The catalytic site is a pocket which is 330Å in volume and a deep of 10Å, with a narrow entry. There are 4 regions: M (with 2 metallic ions), H (hydrophobic), Q (for the substrate), L (the lid or “H-Loop” on both N-term and linker domain). M site is surrounded by the helixes α6, 8, 9, 10 and 12. A majority of aliphatic or hydrophobic residues, that creates the hydrophobic pocket [2].
M site contains:
- The Me-1 and Me-2 sites are occupied by metal ions, Zinc within Me-1 and within Me-2, Zinc, Magnesium or Manganese [5],
- The residues His617, Asp654, Asp764, His653 and two H2O (W1 et W2) binding zinc:
+ The crucial Asp764 [3] and the conserved His617 and 653[4], which bind one Zinc ion, are fundamental for the catalytic activity.
+ Zinc is critical for catalytic activity, but it isn't implied in the formation of the hydrophobic pocket. In fact, even if the His617and 653 are lost and so the Zn not bound, a massive addition of Manganese[4] in the medium allows a reactivation of catalysis.
- W2 binds Zn and Mg [24],
- And there are 3 hydrogen bonds between 3 H2O and the conserved resides His657, Asp682 and His685 [24].
Q site contains:
- In particular the conserved residues Gln817, Phe820, Val782 and Tyr612. [24]
- The hydrogen bonds, between Gln817 and 775, Gln775 and Ala767, Gln775 and Trp853, imply an interaction between Gln817 and the cGMP purine. Thus, it improves the specificity for the cGMP, against cAMP. [24]
- And Tyr612, Val782, Leu785 an Phe820 bind the cGMP through this pyrazol ring and π-π interactions between Gln817 and the phenyl ring. [24]
+ So, the conserved hydrophobic residue Tyr 612 is critical in the maintaining of the affinity. [3]
H site:
- In particular the residues Phe786, Ala783, Leu804, Val782. [24]
L site:
- In particular the hydrophobic residues Tyr664, Met816, Ala823, Gly819. [24]
Besides, the kcat of the catalytic fragment decreases 40-fold and 8-fold if the residues His603 and Asp644 are mutated, and so there are important in the catalytic activity [5,3]. Two others residues are significant: the Gln778 which is important for cGMP affinity but have no impact on cAMP affinity H-loop [8] and the conserved Gly659 which is important for substrate affinity and catalytic activity because it determinates H-loop conformation [21] (see below).
H-loop is important in the substrate recognition and the interactions with, it is from 660th to 693th residues. H-loop has the same interactions with cGMP and Sildenafil (cf. Inhibitor) because it's related to its role of substrate binding[21]. But The H-loop is not well understood, because when it's modified, the enzyme's function is practically not modified.[7] But it also may have a role for inhibitor fixation.
Nowadays catalysis mechanism is not well know: there could be a, nucleophile attack of a water molecule on the substrate [6].
Inhibition
In the treatment erection dysfunction, the inhibitors Sildenafil, Vardenafil and Tadalafil are used, like in the pulmonary hypertension[6]. Sildenafil may cure sleeping trouble after a intercontinental travel [11], may help to recover neural liaisons after an injury (the motor function[12] and the sensory motor function[13]) and can be vascular effects.
- PDE5 inhibitors might help physical condition in Duchene muscular dystrophy[14], improve of cognitive function [9]and have antidepressant effect[10] , also they might have an artero[15] and endothelial cell protective effect[16] so they have cardiac protection effect[17] (controversial, cf. clinical trial “RELAX”), finally they slow tumer cell growth (Tadalafil-like)[18]
- There are other PDE5 inhibitors: IBMX, Icarisid II and Udenafil.
- No interaction between the M site and the inhibitors
For the Sildenafil:
- 3 parts (R1: pyrazolopyrimidinone group; R2: ethoxyphenyl; R3: methylpiperazine)
- It is bound near Metallic ions but does not interact with them.
Binding amino acid for the Sildenafil:
- R1 group have contacts with Gln817 (2 hydrogen bounds, so it increases Sildenafil affinity), Phe820 (Sildenafil stacks against it), Try612 (hydrogen bound so it increases Sildenafil affinity [24]), Leu765 and Ala767 [21]. And there is hydrophobic interactions between the pyrazol ring and residues Val782, Leu785, Tyr612 and Phe820 [24].
- R2 group is in the H pocket and has Van der Waals bounds with Val 782, Ala 783, Phe 786, Leu 804, Ile 813, Gln 817, Phe801. And interaction Pi-Pi between the phenyl ring and the Phe820.
- R3 group is in the L pocket and has contacts with Asn 662, Ser 663, Tyr 664, Ile 665 (in the H-loop), Leu 804, Phe 801
- Gly659 is modified by Sildenafil presence in PDE5, ϕ and φ angles are increased (from 76-105° to 104-109° for ϕ and from 3-22° to 139-141° for φ) and ω angle is not changed. [21]
H-loop:
For each inhibitor, H-loop take a different and originally (comparatively to other PDEs) tertiary structure (and there are also minor modifications of the N-loop (788-811) ):
- For an unliganded PDE5, H-loop take a coil conformation. [21]
- In case of Sildenafil binding, a turn and an 310 helix (from 672 to 675) appear, and residues from 668 to 676 are disordered. The all loop cover the active site (by migrate of 24 Å from unliganded PDE5 loop structure, so the active site become a closed pocket). [21]
- H-loop is less important in the interactions for Sildenafil and Icarisid II than cGMP.
Regulation
As it is written over, there are 2 regulatory domains (GAF A and GAF B). In cGMP pathway, PDE5 allows a negative feedback of the molecule: first, in presence of cGMP, it binds GAF A which stimulates the catalysis in the active site, and vice versa. Moreover, cGMP actives PKG which phophorylates PDE5, that is stimulated by the presence of cGMP on the GAF A or/and the active site. If the protein is not binding with cGMP but it is phophorylated, that stimulates the binding of cGMP on GAF A and the catalytic site. So cGMP presence overstimulates the catalysis [19]. And it also increase inhibitor's affinity[20] and without cGMP, inhibitor don’t bind the PDE5 [22].
The NO Pathway
In the penile erection example, the nervous cell and/or epithelial cells are produced Nitrogen Oxide (NO) by the NOS (NO synthetase) from L-arginine and O2. They release NO in the extracellular environment going into vascular smooth cells and binding the Guanylyl Cyclase. This enzyme synthesizes cGMP from GMP, which stimulates the PKG. Finally, the calcium level is lower and the muscle cell relaxes and the Corpus Cavernosum rigidity increases. The PDE5 regulates the cGMP level making a negative feedback and can stop the rigidity. [22]
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