 |
INTRODUCTION |
cGMP phosphodiesterases
(PDE6)1 play the role of
effector enzymes in the vertebrate visual transduction cascade. In
retinal rod cells, photoexcited rhodopsin induces GDP/GTP exchange on the visual G protein, transducin (Gt), and liberated Gt
GTP activates PDE6. A homologous cascade operates in cone photoreceptors. cGMP hydrolysis by active PDE6 results in the closure of cGMP-gated channels
in the plasma membrane (1, 2). The key attributes of the visual
cascade, low noise and high gain signal amplification, place specific
requirements on PDE6. The enzyme must have a very low basal cGMP
hydrolytic rate in the dark-adapted photoreceptors and a very high
catalytic rate in the transducin-activated state. This is achieved
through two unique features of PDE6: the inhibitory interaction of the
catalytic subunits with the
-subunit and an exceptionally high
kcat value for cGMP hydrolysis when the
inhibition is turned off.
The lack of a practical expression system for PDE6 (3-5) has stalled
the progress in determining the structural basis of PDE6 function. We
have begun to study the structure and function relationship of PDE6 by
constructing chimeras between cone PDE6
' and cGMP binding
cGMP-specific PDE (PDE5 family) (5, 6). PDE5 and PDE6 display a high
degree of identity (45-48%) between the catalytic domains, a strong
substrate selectivity for cGMP, and similar sensitivity to a common set
of competitive inhibitors (7-9). Yet, the reported maximal rate of
cGMP hydrolysis by PDE5 catalytic dimers is only ~10 moles of
cGMP per mole of PDE·sec, which is ~400-550-fold lower than the
kcat estimates for PDE6 (5, 10-15).
Furthermore, the activity of PDE5 is unaffected by the PDE6
-subunit
(5, 6). This, and a robust functional expression of PDE5 using the
baculovirus/insect cell system (16), makes PDE5 a valuable tool for
"gain of PDE6 function" experiments. Recently, we have shown that a
substitution of the segment PDE5-(773-820) by the corresponding
PDE6
'-(737-784) sequence in the wild-type PDE5 or in a
PDE5/PDE6
' chimera containing the catalytic domain of PDE5 results
in chimeric enzymes capable of inhibitory interaction with P
(6).
Alanine-scanning mutational analysis of the previously identified P
cross-linking site, PDE6
'-(750-760) (17), revealed a critical
P
-interacting residue, Met758 (6). In a model of the
PDE6
' catalytic domain, Met758 faces the opening of the
catalytic cavity (6). We then hypothesized that P
may interact with
additional nonconserved residues located at the perimeter of the
cavity, thus allowing P
to serve as a lid on the catalytic pocket.
In this study, we mutated three candidate P
contact residues
identified from the model of PDE6
' and examined these mutants for
inhibition by P
.
The rationale for our search of the catalytic determinants of PDE6 was
based on biochemical evidence and the crystal structure of the PDE4
catalytic domain (18-20), which suggests the critical role of the two
highly conserved metal binding motifs,
His-Asn-X-X-His (I) and
His-Asp-X-X-His (II), in the hydrolysis of cyclic
nucleotides. We replaced PDE6
' domains containing motifs I and II
into PDE5. Resulting chimeric PDEs and corresponding mutants have been
analyzed to test our hypothesis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
cGMP was obtained from Roche Molecular
Biochemicals. [3H]cGMP was a product of Amersham
Pharmacia Biotech. All restriction enzymes were purchased from New
England Biolabs. AmpliTaq® DNA polymerase was a product of PerkinElmer
Life Sciences, and Pfu DNA polymerase was a product of Stratagene.
Rabbit polyclonal His-probe (H-15) antibodies were purchased from Santa
Cruz Biotechnology. Zaprinast and all other reagents were purchased
from Sigma.
Cloning of P
Mutants--
P
mutants were generated based
on the pET11a-P
expression vector (21, 22). Residues
Ile86 and Ile87 were substituted for alanine
using PCR-directed mutagenesis. PCR products were obtained using a
forward primer containing a NdeI site and a reverse primer
containing the mutations and a BamHI site. The fragments
were digested with NdeI/BamHI and subcloned into
the pET11a-P
digested with the same enzymes.
Preparation of P
and P
Mutants--
The P
-subunit and
its mutants were expressed in Escherichia coli and purified
on a SP-Sepharose fast flow column and on a C4 HPLC column
(Microsorb-MW, Rainin) as described (22). Purified proteins were
lyophilized, dissolved in 20 mM HEPES buffer, pH 7.5 and
stored at
80 °C until use.
Cloning of Chi20 and Chi21--
The constructs for expression of
PDE5/PDE6
' chimeras were obtained based on pFastBacHTb-PDE5 vector
(5). To obtain Chi20 and Chi21, original restriction sites in
pFastBacHTb-PDE5, SpeI and SphI, were eliminated
and re-introduced at desired positions to allow a site-directed cloning
of PDE6
' fragments into PDE5. To eliminate two SpeI
restriction sites located within the 3'-untranslated region of PDE5
cDNA and the unique SphI site from the multiple cloning
sequence of the vector, pFastBacHTb-PDE5 was digested with
SpeI/SphI and treated with mung bean nuclease.
New SpeI and SphI restriction sites (PDE5 codons
for Arg606-His607-Ala608 and
Ala618-Leu619-Lys620, respectively)
were introduced into the vector using a QuikChangeTM kit
(Stratagene). To obtain Chi21 (Fig. 1), a synthetic olygonucleotide duplex, encoding for PDE6
'-(561-574), was ligated into the modified pFastBacHTb-PDE5 vector digested with SpeI and
SphI. To generate Chi20, a PCR fragment, encoding for
PDE6
'-(575-617), was digested with SpeI/BlpI
and subcloned into the modified pFastBacHTb-PDE5 vector digested with
SpeI/BlpI(partial). The resulting construct was
digested with SpeI/SphI and ligated to the
synthetic oligonucleotide duplex encoding for PDE6
'-(561-574).
Site-directed Mutagenesis of PDE5 and Chi16--
Site-directed
mutagenesis of PDE5 was performed using a QuikChangeTM kit.
A pair of complementary oligonucleotides encoding for the Ala608
Gly and Ala612
Gly substitutions
(PDE5A608G/A612G) was used to PCR-amplify the pFastBacHTb-PDE5 vector.
The PCR product was treated with DpnI to eliminate the
template and was transformed into E. coli DH5
. Chi16
mutants with single substitutions of residues Lys769,
Phe777, and Phe781 by Ala were constructed
using PCR-directed mutagenesis. A unique NheI site (PDE5
codons for Pro661-Leu662) was introduced into
Chi16 using a QuikChangeTM kit. The 5'-primer sequence
included the NheI recognition site. Reverse primers
contained a desired mutation and the StuI site. The PCR
products were digested with NheI/StuI and
subcloned into the modified Chi16 vector cut with the same enzymes.
Sequences of all mutants were verified by automated DNA sequencing at
the University of Iowa DNA Core Facility.
Expression and Purification of Recombinant PDEs and their
Mutants--
Sf9 cells were harvested at 60 h after
infection, washed with 20 mM Tris-HCl buffer, pH 7.8 containing 50 mM NaCl, and resuspended in the same buffer
containing a protease inhibitor mixture (10 µg/ml pepstatin, 5 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride). The cell suspensions were sonicated using 30-s pulses for a
total duration of 3 min. The supernatants (100,000 × g, 45 min) were loaded onto a column with a His-Bind resin
(Novagen) equilibrated with 20 mM Tris-HCl buffer, pH 7.8, containing 10 mM imidazole. The resin was washed with a 5×
volume of the buffer containing 500 mM NaCl and 25 mM imidazole. Proteins were eluted with the buffer
containing 250 mM imidazole.
-mercaptoethanol (2 mM) was added to the eluate. PDE5, Chi20, Chi21, and
PDE5A608G/A612G were additionally purified using ion-exchange
chromatography on a Mono Q® HR 5/5 column (Amersham
Pharmacia Biotech). Purified proteins were dialyzed against 40%
glycerol and stored at
20 °C.
Other Methods--
PDE activity was measured using
[3H]cGMP as described (23, 24). Less than 15% of cGMP
was hydrolyzed during these reactions. The Ki values
for inhibition of PDE activity by P
and zaprinast were measured
using 0.5 µM cGMP (i.e. <35% of the
Km value for chimeric and mutant PDEs). Protein
concentrations were determined by the method of Bradford (25) using IgG
as a standard or by using calculated extinction coefficients at 280 nm.
The molar concentrations of Chi20, Chi21, and mutatnt PDEs, [PDE], were calculated based on the fraction of PDE protein in preparations, and the molecular mass of 93.0 kDa. The fractional concentrations of
PDE were determined from analysis of the Coomassie Blue-stained SDS
gels using a HP ScanJet II CX/T scanner and Scion Image Beta 4.02 software. A typical fraction of Chi16 mutants in partially purified
preparations was 10-15%. A typical fraction of purified Chi20, Chi21,
and PDE5A608G/A612G was 65-70%. The kcat
values for cGMP hydrolysis were calculated as
Vmax/[PDE]. SDS-polyacrylamide gel
electrophoresis was performed by the method of Laemmli (26) in 10-12%
acrylamide gels. For Western immunoblotting, proteins were transferred
to nitrocellulose (0.1 µm, Schleicher & Schuell) and analyzed using
rabbit His-probe (H-15) or sheep anti-PDE6
' antibodies (5, 6, 27).
The antibody-antigen complexes were detected using anti-rabbit or
anti-goat/sheep IgG conjugated to horseradish peroxidase and ECL
reagent (Amersham Pharmacia Biotech.). Fitting the experimental data to
equations was performed with nonlinear least squares criteria using
GraphPad Prizm Software. The Ki,
Km, and IC50 values are expressed as mean ± S.E. for three independent measurements.
 |
RESULTS |
Mutational Analysis of the P
Binding Site of
PDE6
'--
Previously, we demonstrated that PDE5/PDE6
' chimeras
containing a PDE6
' sequence, PDE6
'-(737-784), are effectively
inhibited by P
, and two residues, Met758 and
Gln752, participate in the inhibitory interaction (6).
Based on the model structure of PDE6
' (6), three solvent-exposed
nonconserved PDE6
' residues, Lys769, Phe777,
and Phe781, were chosen for further mutational analysis of
the P
binding region (Fig.
1A). A PDE5/PDE6
' chimera,
Chi16 (6), served as a template for single substitutions of these
residues by Ala. The Chi16 mutants were expressed in Sf9 insect
cells and partially purified. Expression of the K769A, F777A, and F781A
mutants have yielded similar amounts of soluble protein (50-100
µg/100 ml of culture). Neither of these mutations has significantly
affected the catalytic properties of chimeric PDE. The
Km and kcat values for cGMP
hydrolysis for all three mutants were in the 3-10 µM
range, and the 5-10 s
1 range, respectively (Table
I). As an additional control for the
structural integrity of the catalytic site, mutants of Chi16 were
tested for the PDE activity inhibition by zaprinast, a specific competitive inhibitor of PDE5 and PDE6. The largest change, a 2-fold
increase in the IC50 value, was caused by the F781A
substitution (Table I). Nonetheless, such a change represents an
insignificant loss of affinity to zaprinast.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Construction and expression of
PDE5/PDE6 ' chimeras and mutants in Sf9
cells. A, schematic representation of PDE5/PDE6 '
chimeras. The PDE6 ' residues substituted by Ala in Chi16 and the
metal binding motifs I and II are shown. The PDE6 and PDE5 motifs I are
identical. B, an SDS-polyacrylamide gel (12%) of purified
PDE5, Chi20, Chi21, and PDE5A608G/A612G (2 µg per lane) stained with
Coomassie Blue. The recombinant His6-tagged proteins were
expressed in Sf9 cells and partially purified using
chromatography on a His-Bind resin and HPLC on a Mono Q®
HR 5/5 column as described under "Experimental Procedures."
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Functional properties of PDE5/PDE6 ' chimeras
PDE activity was measured using [3H]cGMP (24). The
Km values of PDE6 ' or PDE5 and PDE5/PDE6 '
chimeras were determined in the presence of 0.1 µCi
[3H]cGMP and 0.1-500 µM of unlabeled cGMP. The
Ki and IC50 values for inhibition of PDE
activity by P and zaprinast were measured using 0.5 µM
cGMP. The results are presented as the mean ± S.E. for three
independent measurements.
|
|
The test of the ability of Chi16 mutants to be inhibited by P
showed
that the K769A mutation had no effect on the inhibitory interaction
with P
(Ki 2.9 nM) (Table I). Two
other mutants, F777A and F781A, displayed significant impairments in the inhibition by P
. The F777A substitution reduced both the maximal
inhibition of PDE activity by P
(~45%) and the
Ki value (Ki of 19 nM). The inhibition of F781A mutant by P
also was
incomplete (~65%) and associated with an increase in the
Ki value (Ki of 31 nM) (Fig. 2A and
Table I).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of P and
C-terminal P mutants on the catalytic activity
of tPDE6 ' Chi16 and Chi16 mutants.
A, inhibition of Chi16, F777A, and F781A PDE activity by
P . The activities of Chi16 ( ), F777A ( ), and F781A ( )
(50-100 pM) were determined upon addition of increasing
concentrations of P . Reactions were initiated by addition of 0.5 µM of cGMP. The Ki values (nM)
calculated from the inhibition curves were 3.6 ± 0.4 ( ),
19 ± 2 ( ), and 31 ± 5 ( ). B, inhibition of
tPDE6 ' ( ) (0.5 pM), Chi16 ( ), F777A ( ), and
F781A ( ) by P I86A. The Ki values (nM)
calculated from the inhibition curves were 0.75 ± 0.08 ( ),
13 ± 1 ( ), 96 ± 13 ( ), and 49 ± 8 ( ).
C, inhibition of tPDE6 ' ( ), Chi16 ( ), F777A ( ),
and F781A ( ) by P I87A. The Ki values
(nM) calculated from the inhibition curves were 0.65 ± 0.04 ( ), 6.6 ± 1.0 ( ), 64 ± 8 ( ), and 32 ± 2 nM ( ).
|
|
Effects of the C-terminal P
Mutants on the Catalytic Activity of
Mutant Chi16--
C-terminal P
mutants were designed based on the
evidence for the critical role of the P
C terminus in PDE6
inhibition (21, 28). The two extreme C-terminal P
residues,
Ile86 and Ile87, were replaced by Ala to obtain
the P
I86A and P
I87A mutants, respectively. The P
mutants were
analyzed for their ability to inhibit trypsin-activated PDE6
'
(tPDE), Chi16, and the M758A, F777A, and F781A mutants (Fig. 2; Table
I). P
I86A and P
I87A fully inhibited tPDE activity. However, the
potency of the inhibition was reduced ~4-5-fold
(Ki of 0.75 nM for P
I86A and
Ki of 0.65 nM for P
I87A, compared
with Ki of 0.15-0.2 nM for P
). A
similar increase in the Ki values was observed from the inhibition of Chi16 activity by P
I86A (Ki of
13 nM) and P
I87A (Ki of 7 nM) (Fig. 2, B and C; Table I). Yet,
P
I86A and P
I87A did not fully inhibit Chi16, maximal inhibition was 65 and 70%, respectively. (Fig. 2, B and C;
Table I). No appreciable inhibition of M758A by either P
mutant was
seen even at inhibitor concentrations as high as 5 µM.
The inhibition of F777A by P
I86A was partial (45%) with the
Ki value of 96 nM, whereas P
I87A
inhibited this Chi16 mutant with an even smaller maximal effect (25%,
Ki of 64 nM). The F781A mutant was
inhibited by P
I86A and P
I87A with Ki
values of 49 and 32 nM and maximal effects of 40 and 55%,
respectively (Fig. 2, B and C; Table I).
Catalytic Properties of PDE5/PDE6
' Chimeras Containing the
PDE6
' Metal Binding Sites--
Two conserved metal binding motifs
found in all PDEs are absolutely critical for cyclic nucleotide
hydrolytic activity (18-20). To identify the structural elements
responsible for the unique catalytic properties of PDE6, chimeric
PDE5/PDE6
' have been generated by introduction into PDE5 of PDE6
'
domains containing metal binding motifs, I and II. A replacement of the
PDE6
'-(562-617) segment into PDE5 yields a chimeric PDE5/PDE6
',
Chi20, that incorporates both PDE6
' metal binding sites and the
connecting sequence (Fig. 1A). Chi20 was expressed in
Sf9 cells as a functional enzyme at ~400 µg/100 ml and
purified to ~ 65-70% purity (Fig. 1B). The catalytic characteristics of Chi20 were examined in comparison to those
of PDE5 and native PDE6
'. PDE6
' has reported
Km (17-25 µM) and
kcat (3500-4500 moles of cGMP per mole of
PDE·s) values for cGMP hydrolysis that are ~5 and ~400-fold
higher than the respective constants for PDE5 (5, 10-11, 14). The
catalytic parameters of Chi20 were significantly different from those
of PDE5. Chi20 hydrolyzed cGMP with the Km value of
12 µM, which is ~4-fold higher than the
Km value for PDE5 but similar to that of PDE6
'
(Table I). The maximal activity of 116 moles of cGMP per mole of
PDE·s for Chi20 is ~10-fold higher than that of PDE5. Chi20 was
inhibited by zaprinast with the IC50 value of 0.35 µM, which is comparable with that of PDE5 (Table I).
To determine the role of individual metal binding motifs and their
adjacent regions in cGMP hydrolysis by PDE6, we inserted a PDE6
'
fragment corresponding to the helix-
6 (20), PDE6
'-(562-574), into PDE5 (Chi21) (Fig. 1). The catalytic properties of Chi21 and the
inhibition by zaprinast (Km of 17 µM,
kcat of 110 moles of cGMP per mole of PDE·s,
and IC50 0.39 µM) were similar to those of Chi20.
Catalytic Properties of the PDE5A608G/A612G Mutant--
The
alignment of sequences from different PDE families corresponding to the
6 helix shows a glycine residue, PDE6
'Gly562,
conserved only in photoreceptor PDEs (Fig.
3A). A second Gly residue,
PDE6
'Gly566, is conserved in PDE6
' and PDE6
, but
substituted by Ala in PDE6
and PDE5 (Fig. 3A). To test
the hypothesis that Gly562 and Gly566 of
PDE6
' are responsible for the differences in catalytic properties of
Chi21 and PDE5, a doubly substituted mutant of PDE5, A608G and A612G,
was expressed and purified from Sf9 cells. Similar to Chi20 and
Chi21, PDE5A608G/A612G hydrolyzed cGMP with a Km value of 14 µM and a kcat value of
105 moles of cGMP per mole of PDE·s (Table I).

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 3.
The P C terminus
docked to the PDE6 ' catalytic site.
A, an alignment (31) of PDE1-6 sequences corresponding to
the helix- 6 (20). B and C, a model of
the PDE6 ' was generated with SWISS-MODEL (32) using the coordinates
of the PDE4 structure as a template (20). The P C terminus binding
residues, Gln752, Met758, Phe777,
and Phe781 are shown in red. The metal ions
Zn2+ and Mg2+ are shown in yellow
and magenta, respectively. The C terminus of P ,
P -(75-87), was generated and manually docked to the catalytic site
using SYBYL (v.6.7) (Tripos Associates, St. Louis, MO). C,
the clipped view is a 900 counterclockwise rotation around
the vertical axis shown in B.
|
|
 |
DISCUSSION |
An interaction between PDE6 catalytic and inhibitory P
-subunits
keeps the visual effector enzyme inhibited in the dark. Previous biochemical studies have established that the
-subunit of
photoreceptor PDE inhibits the enzyme activity by blocking its
catalytic site (29). The major inhibitory domain has been localized to
the P
C terminus (21, 28). Recently, we have demonstrated that P
inhibits the activity of PDE5/PDE6
' chimera, Chi 16, containing residues PDE6
'-(737-784) (6). Essential P
binding residues, Gln752 and Met758, of PDE
' have been
identified via mutagenesis of Chi16 (6). A model of the PDE6
'
catalytic domain places Met758 at the opening of the
catalytic pocket (6). Hypothetically, to ensure an effective catalytic
block, the P
C terminus may lie over or might be inserted into the
catalytic cavity. The former appears more likely because the catalytic
pockets of different cyclic nucleotide PDEs are made up of highly
conserved residues, whereas the inhibition by P
is a unique
attribute of PDE6. We speculated that to cover the catalytic pocket,
the P
C terminus, besides Met758, interacts with
additional nonconserved residues located at the perimeter of the
entrance to the active site. The fact that the introduction of
PDE6
'-(737-784) into PDE5/PDE6
' chimera leads to a full
inhibition of the PDE activity by P
suggests the PDE6
'-(737-784) segment contains most if not all residues interacting with the P
C
terminus. In the PDE6
' model, PDE6
'-(737-784) comprises about
half of the catalytic cavity mouth. Residues at three positions within
PDE6
'-(737-784) (Lys769, Phe777, and
Phe781) are conserved among photoreceptor PDEs but have
nonhomologous substitutions in PDE5. Supporting our hypothesis,
replacement of two residues, Phe777 and Phe781,
by Ala in Chi16 has resulted in mutant PDEs that in comparison with
Chi16 were less potently and incompletely inhibited by P
. Phe777 and Phe781 are located next to each
other, opposite to the Met758 side of the catalytic opening
(Fig. 3, B and C). Thus, it appears that the P
C terminus makes a bridge over the catalytic pocket. Such a model
provides an interesting explanation to the results of an earlier study
that examined inhibition of PDE6 by C-terminally truncated P
mutants
(21). Truncations of one or two of the C-terminal
Ile86-Ile87 residues led to substantial
increases in the Ki value, whereas further
truncations, up to 8-11 C-terminal residues, reduced the maximal
inhibition of PDE6 activity without significantly affecting the
Ki value (21). A plausible interpretation is that
P
Ile86-Ile87 interact with residues on one
side of the catalytic pocket and other residues, perhaps P
-(77-85),
stretch over the catalytic cavity until P
reaches the opposite side.
Accordingly, removal of P
Ile86-Ile87
decreases the affinity of P
for the PDE6 catalytic subunit, whereas
progressive removal of P
-(77-85) residues gradually facilitates access of cGMP to the catalytic site. To determine the orientation of
the P
C terminus against the catalytic site and identify
point-to-point interactions with PDE6
', we examined the inhibition
of Chi16 and the M758A, F777A, and F781A mutants of Chi16 by two P
mutants, P
I86A and P
I87A. The simplest prediction is that if a
C-terminal Ile of P
interacts with one of the three PDE6
'
residues, the corresponding mutant PDE would be inhibited comparably by
P
and by the P
mutant. Complicating this prediction, side chains
of Phe777 and Phe781 make a hydrophobic contact
and thereby may support each other in the interaction with P
. The
analysis of inhibition of Chi16 mutants by P
mutants indicates that
Ile86 and Ile87 of P
interact with
Phe777 and Phe781 of PDE6
'. Moderate
increases in the Ki values and reductions in the
maximal inhibition of F777A and F781A caused by the P
I86A substitution suggest that Ile86 probably contacts one or
both the PDE6
' residues. The failure of P
I86A to inhibit M758A is
consistent with the notion that Ile86 binds
Phe777/781, but not Met758. The lack of
inhibition is likely caused by the inability of M758A and P
I86A to
establish at least two of the three critical contacts involving
Met758, Phe777, and Phe781. The
P
I87A mutant did not appreciably inhibit the activity of the M758A
mutant PDE. P
I87A inhibited F781A stronger than F777A pointing to a
probable contact between P
Ile87 and Phe781
of PDE6
'. The incomplete inhibition of mutant PDEs by P
or P
mutants most likely reflects equivalent partial inhibition of both
active sites of the catalytic dimer, rather than the loss of inhibition
at one site.
The analysis of P
secondary structure predicts an
-helical
structure for the C-terminal residues P
-(75-84) (30). The C
terminus of P
, P
-(75-87), manually docked to the PDE6
'
catalytic site is shown in Fig. 3, B and C. The
model assumes the helical structure of P
-(75-84) and the contacts
between P
Ile86-Ile87 and
PDE6
'Phe777-Phe781. This orientation of P
is also consistent with Gln752 of PDE6
' (6) making a
contact with a P
residue located N-terminally to P
-(75-87).
The remarkable ability of photoreceptor PDEs to hydrolyze cGMP with a
catalytic rate constant of ~4000-5500 moles of cGMP per mole of
PDE·s (12-15) is essential to the signal amplification in the visual
cascade. All catalytic subunits of cyclic nucleotide PDEs contain two
strictly conserved metal binding motifs,
His-Asn-X-X-His (motif I) and
His-Asp-X-X-His (motif II). In PDE6
' these
motifs are as follows:
557His-Asn-Trp-Arg-His561 and
597His-Asp-Ile-Asp-His601. The crucial role of
the metal ions and the binding motifs for PDE catalytic activity has
been recently supported by a crystallographic study of the PDE4
catalytic domain (20). Rather than forming separate metal binding
sites, both motifs are involved in coordination of two bound metal
ions, ME1 and ME2 (20). For example, ME1, most likely a tightly bound
Zn2+, is coordinated by the His residue (His561
of PDE6
') from motif I, and the His and Asp residues from motif II
(His597-Asp598). A model of cAMP docked in the
PDE4 active site demonstrates that ME1 and ME2 bind the cyclic
phosphate, position a potential water molecule for the nucleophilic
attack, and would serve to stabilize the transition state (20). In view
of the role of metal binding sites in hydrolysis of cyclic nucleotides,
we have considered the motifs I and II as probable structural
determinants of the catalytic properties of PDE6. Motifs I and II are
practically identical in PDE5 and PDE6. Therefore, a spatial
orientation of these sites might be a potential key factor for cGMP
hydrolysis. Motif I comprises the N-terminal potion of the helix-
6,
and motif II is in the loop connecting helices 7 and 8. A PDE5/PDE6
'
chimera, Chi20, was generated by replacing a PDE6
' domain containing
helices
6-
8 into PDE5. The analysis of Chi20 revealed a more than
10-fold increase in the maximal catalytic rate accompanied by a
~5-fold increase in the Km value. Subsequent
chimeric PDE, Chi21, containing only helix
6 of PDE6
' displayed
catalytic properties similar to those of Chi20. An alignment of
sequences of photoreceptor PDEs and PDE5 corresponding to the
helix-
6 shows a high degree of homology with the notable exception
of residues at two positions corresponding to PDE6
'
Gly562 and Gly566. Gly562 of
PDE6
' is conserved only in the PDE6 family, but substituted by Ala
in PDE5 (Fig. 3A). Importantly, Gly562
immediately follows His561 from motif I. His561, by analogy to PDE4, is involved in coordination of
ME1, and in the positioning of His557 to accomplish the
protonation of the O3' leaving group (20). To probe the role of the Gly
residues, a doubly substituted PDE5 mutant, A608G/A612G, has been made.
The kcat value of the A608G/A612G mutant was
comparable with those of Chi20 and Chi21, and ~10-fold higher then
that of PDE5. These results suggest that the Gly residues are in part
responsible for the catalytic characteristics of PDE6. Most likely,
they allow for a positioning of motif I that is most favorable for cGMP
hydrolysis. Other yet to be defined determinants contribute to the
unique catalytic power of PDE6, because the achieved
kcat value is still ~40-50-fold lower than
kcat described for native activated PDE6.
Overall, our results suggest that a progressive incorporation of PDE6
domains or residues into PDE5 not only allows a structure-function
analysis of PDE6, but also represents a realistic approach to generate
a chimeric enzyme that would be functionally indistinguishable from PDE6.