Cyclic nucleotide specificity

The six member phosphodiester ring and the purine moiety of c AMP and cGMP are critical for interaction with the PDEs as other nucleotides (ATP, ADP, GTP, GDP) do not interact

with PDEs. cAMP and cGMP are also resistant to other phosphodiesterases such as intestinal phosphatases. PDEs can be very selective for one or the other o f the cyclic

nucleotides as with cAMP-speciflc PDE4, PDE7 and PDE8 which have values of 1 -

4p,M for cAMP while having values greater than 1 OOOpM for cGMP. PDE5, PDE6 and

PDE9 have a preference for cGMP while PDEl, PDE2, PDE3, PDE 10 and PDEl 1 have

comparable values for both cyclic nucleotides. This cyclic nucleotide selectivity of the

catalytic domains is determined by interactions between residues or sequence of residues within the catalytic domain and the substrate (Turko et a l , 1996; Turko et a l, 1998a). Site- directed mutagenesis studies on cGMP-specific PDE5 have revealed a short sequence of -30 amino acid residues around an invariant glutamic acid (Glu^^^) as being important for substrate specificity (Turko et al, 1996; Turko et al, 1998a). Turko and co-workers (1998a) showed that by replacing residues around this Glu^^^ with conserved residues in the corresponding positions of c AMP-specific PDEs, the substrate affinity o f the enzyme could be altered (amino acid residues shown in colour in Figure 1.10). They found that when particular residues were mutated, there was a lowering o f the PDE5 affinity for cGMP. The most significant effect seen was a substantial reduction in substrate affinity when Glu(E)^^^

and Tyr(Y)^®^ were replaced with alanine (A). This increased the for cGMP from 2pM

for the wild-type to 70p,M for E775A mutation, and 65p,M for the Y602A mutation. Their experiments also revealed that by substituting just two residues of the PDE5 enzyme (Trp^^^ and Gln^^^) with the residues in the corresponding position of PDE4 (Leu and Tyr respectively), the cGMP/cAMP selectivity could be shifted by -80-fbld in favour o f cAMP hydrolysis (Table 1.3, Turko et al, 1998b). Figure 1.10 shows the sequence alignment of the amino acid residues for PDE5A1, PDE4 and PDEl enzymes corresponding to the section around the invariant Glu^^^ for PDE5. It includes the corresponding sequence for the dog heart PD ElA l (AF252536) used in the present study for comparison. Since this portion of -3 0 amino acids is conserved to a large extent between the PDE families, it is possible that site-directed mutagenesis experiments on other PDEs would give similar shifts in substrate affinities. However, further experiments by Francis and co-workers (2000b) revealed that catalytic activity in some of the mutants could be restored by the addition of divalent metal ions and that the results obtained in the mutagenesis experiments apply under the conditions used for these studies (Francis e/ûf/., 2000b). Despite this dependence on assay conditions for the mutagenesis experiment results, the importance of the residues

workers following their elucidation of the crystal structure of PDE4B2B catalytic domain (2000). Residues interacting with cAMP are discussed in section 1.4.7.4.4 and are highlighted in Figure 1.14.1.

BTPDE5A1

BTPDElAl

CFPDElAl

HSPDElAl

HSPDE4B2B

7 6 2 765

I I

770

I

775 I ''“ DLSAITKPWPIQQRIAELVATEFFDQGDRE^®^' ^^^DISHPAKSWKLHHRWTMALMEEFFLQGDKE^^^ ^^“DISHPAKSWQLHYRWTMALMEEFFLQGDKE” ^ DISHPAKSWKLHYRWTMALMEE FFLQGDKE ^^^DLSNPTKSLELYRQWTDRIMEEFFQQGDKE^^' À À À A À k k k 3 9 2 3 9 4 398 4 0 0 4 0 3 _{4 1 3 4 1 4 4 1 7 4 1 8 421}

Figure 1.10 Amino acid sequence alignment for PDE5, PDE4 and PDEl enzymes showing conserved residues (shown coloured) around the invariant glutamic acid (shown underlined). T h e c o l o u r e d r e s i d u e s in b o v i n e P D E 5 A I ( b o l d ) r e p r e s e n t t h e r e s i d u e s m u t a t e d b y T u r k o a n d c o - w o r k e r s ( 1 9 9 6 , 1 9 9 8 a ) a n d s h o w n to b e i m p o r t a n t in c G M P s p e c i f i c i t y . P o s i t i o n s o f t h e s p e c i f i c a m i n o a c i d s r e f e r r e d to in t h e t e x t w i t h r e f e r e n c e t o P D E 5 a r e i n d i c a t e d b y t h e a r r o w s a b o v e t h e P D E 5 s e q u e n c e E^ ’ h - R e s i d u e s h i g h l i g h t e d f o r H S P D E 4 B 2 B ( a r r o w - h e a d s b e l o w t h e s e q u e n c e ) a r e a l s o h i g h l i g h t e d in t h e r i b b o n d i a g r a m ( F i g u r e 1 . 1 3 . 2 ) o f t h e c a t a l y t i c d o m a i n o f P D E 4 B 2 B . S e q u e n c e s s h o w n a r e B T P D E 1 A 5 ( M e A l l i s t e r - L u c a s e t a l ., 1 9 9 3 ) , B T P D E l A l ( S o n n e n b u r g e t a l. 1 9 9 5 ) , C F P D E l A l ( G e n B a n k A c c e s s i o n n u m b e r A F 2 5 2 5 3 6 ) , H S P D E l A l ( G e n B a n k A c c e s s i o n n u m b e r A L 1 1 0 2 6 3 ) a n d H S P D E 4 B 2 B ( G e n B a n k A c c e s s i o n n u m b e r L 2 0 9 7 1 ) .

Table 1.3 cGMP selectivity of PDE5A1 following site-directed mutagenesis (Turko g/a/., 1998a, 1998b). P D E 5 A 1 c G M P c A M P Wild type 2 Y602A 65 Y6021 2 T713A 30 D 7I4A 5 E775A 70 F775D 6

A769T/1 7 7 1 R (double mutant) 8

W762L./0765 Y (double mutant) 36

W 762L/Q765Y/A769T/L771 R (m ultiple m utant) 43

330 NT NT NT NT NT NT 84 77 67 Simplistie elassifieation o f amino aeid residues:

Hydrophobic ammo acid residues: Ala. A lie. I Leu. I Rhc. I (am m atict

Hydrophilic ammo acid residues: Arg, R Asp, D Glu, H Gin, Q Lys, K Tyr, Y (arom atic) Thr, T Trp, W (aromatic)

Hydropathy analysis was also carried out by Turko and co-workers (1998b) on selected PDEs to compare selected sequences around the invariant Glu^^\ These revealed that the amino acid residues around this Glu^^^ in the cGMP-speeifie PDE enzymes (PDE5 and

PDE6) were predominantly hydrophobie while those in the cAMP-specific PDE enzymes

(PDE4 and PDE7) were predominantly hydrophilie. PDE enzymes with a dual substrate specificity (PDE 1, PDE2 and PDE3) had a mixture of hydrophobie and hydrophilie residues around the invariant Glu. The site directed mutagenesis earried out by Turko and eo- workers on PDE5 enzyme altered the hydrophathy profiles such that while the wild-type enzyme contained predominantly hydrophobic residues around the invariant Glu^^\ the mutated enzymes were made progressively more hydrophilic resembling the hydropathy profile of the cAMP-specific PDE4 enzymes. This reflected in the substrate affinities as a shift from cGMP-speeificity to equal affinity for both cAMP and cGMP as seen by the

values (Table 1.3).

amino acids interacting with cAMP. This will be discussed in a separate section but it is worth noting that the importance of the amino acid residues described as being vital in substrate interactions by Turko and co-workers in their mutagenesis experiments (1998a, 1998b) have been said to be involved with cAMP binding by Xu and co-workers (2000). The stretch of -3 0 amino acids around the invariant in PDE4 (equivalent to the invariant Glu^^^ in PDE5) are contained within helix 14 of the catalytic domain of the PDE4 structure described. Helix 14 forms part o f the deep pocket in the carboxy-subdomain of the catalytic domain and is where the substrate cAMP is accommodated for subsequent hydrolysis by the enzyme. Figure 1.13.1 shows a simple schematic of the subdomain organisation of the catalytic structure described by Xu and co-workers (2000) while Figure

1.13.2 shows the ribbon diagram of the subdomain organisation of the catalytic domain. The position of the amino acids for PDE4B corresponding to the residues mutated in the PDE5 mutagenesis experiments are also highlighted in Figure 1.13.2.

In document Expression and characterisation of a novel cyclic nucleotide phosphodiesterase Type lA from dog heart (Page 44-48)