From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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ABSTRACT |
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The known mammalian 3':5'-cyclic nucleotide
phosphodiesterases (PDEs) contain a conserved region located toward the
carboxyl terminus, which constitutes a catalytic domain. To identify
amino acids that are important for catalysis, we introduced
substitutions at 23 conserved residues within the catalytic domain of
the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5).
Wild-type and mutant proteins were compared with respect to
Km for cGMP, kcat, and
IC50 for zaprinast. The most dramatic decrease in
kcat was seen with H643A and D754A mutants with
the decrease in free energy of binding (GT)
being about 4.5 kcal/mol for each, which is within the range predicted
for loss of a hydrogen bond involving a charged residue.
His643 and Asp754 are conserved in all known
PDEs and are strong candidates to be directly involved in catalysis.
Substitutions of His603, His607,
His647, Glu672, and Asp714 also
produced marked changes in kcat, and these
residues are likely to be important for efficient catalysis. The Y602A
and E775A mutants exhibited the most dramatic increases in
Km for cGMP, with calculated
GT of 2.9 and 2.8 kcal/mol, respectively,
that these two residues are important for cGMP binding in the catalytic
site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the
key residues for its binding differ significantly from those that bind
cGMP.
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INTRODUCTION |
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The 3':5'-cyclic nucleotide phosphodiesterase (PDE)1 superfamily catalyzes the hydrolysis of 3':5'-cyclic nucleotides to the corresponding nucleoside 5'-monophosphates. On the basis of their structural, kinetic, and regulatory characteristics, they have been recently classified into seven major families (1). Comparison of the reported PDE sequences reveals a conserved region of approximately 270 amino acids located toward the COOH terminus of PDE molecules (2). This region is more conserved within an individual PDE family (65-80% amino acid identity) than among different PDE families (25-40% identity). Studies using limited proteolysis of the different PDEs (3-6), deletion mutagenesis (7-9), and point mutations targeting conserved residues (7) strongly support the assertion that this region constitutes a catalytic domain of all PDEs. In addition to the conserved residues that play a role in catalysis and substrate binding, the catalytic domain is likely to contain determinants that confer cyclic nucleotide specificity of different PDEs.
cGMP-binding cGMP-specific PDE (cGB-PDE; PDE5A) is an enzyme with high selectivity for cGMP as substrate. In addition to the site of cGMP hydrolysis, cGB-PDE contains two allosteric cGMP-binding sites that are located toward the NH2 terminus of the cGB-PDE molecule (10). Our ultimate aim is to construct a comprehensive structure-function map of the cGB-PDE using site-directed mutagenesis as a tool. Recently, we replaced several conserved residues in the high affinity allosteric site a (11) and proposed a role of each residue in the putative NKXnD motif, which constitutes a new class of cGMP-binding sites. Detailed analysis of the sequence alignment of the catalytic region of all known PDEs to date reveals two blocks of conserved residues (10). One of these blocks has some sequence similarity to the allosteric binding sites (12), which might suggest some evolutionary relationship between cGMP binding in the allosteric and catalytic sites. However, the cGMP-binding properties and the function of the allosteric sites are quite different from those of the catalytic site. Another block of the conserved residues possesses sequence similarity to Zn2+-binding sites of the different Zn2+-dependent hydrolases, and could be a part of the PDE catalytic mechanism (13).
In the present study, scanning mutagenesis has been used to examine the importance of 23 conserved amino acids in the catalytic domain of the cGB-PDE in maintaining catalytic function. Each of these 23 conserved residues has been substituted individually. After expressing and partially purifying the mutant proteins, we have assessed the effect of these mutations on substrate binding, catalysis, and specific inhibitor binding by measuring the Km value for cGMP, kcat, and IC50 for zaprinast, respectively.
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EXPERIMENTAL PROCEDURES |
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Materials-- [3H]cGMP was purchased from Amersham Corp. cGMP, histone VIII-S, Crotalus atrox snake venom, 3-isobutyl-1-methylxanthine, and zaprinast were obtained from Sigma. Hydroxyapatite was from Bio-Rad.
Site-directed Mutagenesis-- cGB-8/14 clone encodes a full-length bovine lung cGB-PDE (11). The QuikChange site-directed mutagenesis kit (Stratagene) has been used to make point mutations in the cGB-8/14 clone in pBacPAK9 expression vector (CLONTECH) according to the protocol from Stratagene. The following pairs of mutagenic oligonucleotides were used: 1) Y596A, 5'-GT GTG AAG AAG AAC GCT CGG AAG AAC GTC G-3' and 5'-C GAC GTT CTT CCG AGC GTT CTT CTT CAC AC-3'; 2) Y602A, 5'-GG AAG AAC GTC GCC GCT CAT AAT TGG AGA C-3' and 5'-G TCT CCA ATT ATG AGC GGC GAC GTT CTT CC-3'; 3) Y602F, 5'-GG AAG AAC GTC GCC TTT CAT AAT TGG AGA C-3' and 5'-G TCT CCA ATT ATG AAA GGC GAC GTT CTT CC-3'; 4) H603A, 5'-G AAC GTC GCC TAT GCT AAT TGG AGA CAT GCC-3' and 5'-GGC ATG TCT CCA ATT AGC ATA GGC GAC GTT C-3'; 5) N604A, 5'-C GTC GCC TAT CAT GCT TGG AGA CAT GCC-3' and 5'-GGC ATG TCT CCA AGC ATG ATA GGC GAC G-3'; 6) H607A, 5'-GCC TAT CAT AAT TGG AGA GCT GCC TTT AAT ACA GC-3' and 5'-GC TGT ATT AAA GGC AGC TCT CCA ATT ATG ATA GGC-3'; 7) E632A, 5'-GG CTG ACG GAC CTG GCG ATA CTT GCA CTG C-3' and 5'-G CAG TGC AAG TAT CGC CAG GTC CGT CAG CC-3'; 8) H643A, 5'-GCT GCC TTA AGC GCT GAT CTG GAT CAC CGT GG-3' and 5'-CC ACG GTG ATC CAG ATC AGC GCT TAA GGC AGC-3'; 9) D644A, 5'-GCC TTA AGC CAT GCT CTG GAT CAC CGT GG-3' and 5'-CC ACG GTG ATC CAG AGC ATG GCT TAA GGC-3'; 10) H647A, 5'-GC CAT GAT CTG GAT GCC CGT GGT GTC AAT AAC-3' and 5'-GTT ATT GAC ACC ACG GGC ATC CAG ATC ATG GC-3'; 11) E672A, 5'-C CAT TCA ATC ATG GCG CAT CAT CAT TTT G-3' and 5'-C AAA ATG ATG ATG CGC CAT GAT TGA ATG G-3'; 12) H674A, 5'-CA ATC ATG GAG CAT GCT CAT TTT GAT CAG TGC C-3' and 5'-G GCA CTG ATC AAA ATG AGC ATG CTC CAT GAT TG-3'; 13) H675A, 5'-C ATG GAG CAT CAT GCT TTT GAT CAG TGC C-3' and 5'-G GCA CTG ATC AAA AGC ATG ATG CTC CAT G-3'; 14) T713A, 5'-GCT ATT TTA GCC GCA GAC CTA GCA CTG-3' and 5'-CAG TGC TAG GTC TGC GGC TAA AAT AGC-3'; 15) D714A, 5'-GCT ATT TTA GCC ACA GCC CTA GCA CTG-3' and 5'-CAG TGC TAG GGC TGT GGC TAA AAT AGC-3'; 16) D754A, 5'-G ATG ACA GCT TGT GCT CTT TCT GCA ATT AC-3' and 5'-GT AAT TGC AGA AAG AGC ACA AGC TGT CAT C-3'; 17) S756A, 5'-GCT TGT GAT CTT GCT GCA ATT ACA AAA CCC-3' and 5'-GGG TTT TGT AAT TGC AGC AAG ATC ACA AGC-3'; 18) K760M, 5'-CT GCA ATT ACA ATG CCC TGG CCT ATT CAA CAA CGG-3' and 5'-CCG TTG TTG AAT AGG CCA GGG CAT TGT AAT TGC AG-3'; 19) E775A, 5'-CTT GTT GCC ACT GCA TTT TTT GAC CAA GG-3' and 5'-CC TTG GTC AAA AAA TGC AGT GGC AAC AAG-3'; 20) E775D, 5'-CTT GTT GCC ACT GAC TTT TTT GAC CAA GG-3' and 5'-CC TTG GTC AAA AAA GTC AGT GGC AAC AAG-3'; 21) E775Q, 5'-CTT GTT GCC ACT CAA TTT TTT GAC CAA GG-3' and 5'-CC TTG GTC AAA AAA TTG AGT GGC AAC AAG-3'; 22) F776L, 5'-GCA GAA CTT GTT GCC ACT GAA CTT TTT GAC CAA GGA G-3' and 5'-C TCC TTG GTC AAA AAG TTC AGT GGC AAC AAG TTC TGC-3'; 23) Q779A, 5'-GCC ACT GAA TTT TTT GAC GCA GGA GAT AGA GAG AGG-3' and 5'-CCT CTC TCT ATC TCC TGC GTC AAA AAA TTC AGT GGC-3'; 24) G780A, 5'-GCC ACT GAA TTT TTT GAC CAA GCA GAT AGA GAG AGG-3' and 5'-CCT CTC TCT ATC TGC TTG GTC AAA AAA TTC AGT GGC-3'; 25) D781A, 5'-GAC CAA GGA GCT AGA GAG AGG AAA GAA CTC-3' and 5'-GAG TTC TTT CCT CTC TCT AGC TCC TTG GTC-3'; 26) E783A, 5'-GAC CAA GGA GAT AGA GCG AGG AAA GAA CTC-3' and 5'-GAG TTC TTT CCT CGC TCT ATC TCC TTG GTC-3'. The altered bases are underlined. To avoid theoretically possible random mutations, the 1073-bp fragments containing the desired mutations were excised from cGB-8/14 using KpnI/Bst1107I digestion, and resubcloned in the wild-type cGB-8/14 clone in the pBacPAK9 vector using the same restriction sites.
E. coli XL1-blue cells were used for all transformations. DNA fragments were purified by the freeze squeeze method from agarose slices using SPIN-XTM centrifuge filter units (Costar). DNA was purified from large scale vector preparations using a QIAGEN Plasmid Maxi kit according to the manufacturer's protocol (QIAGEN). All DNA segments subjected to mutagenesis, and subcloning reactions, were sequenced in their entirety to ensure the presence of the desired mutation and proper in-frame subcloning.Expression of Wild-type and Mutants-- Sf9 cells were cotransfected with Bsu36I-digested BacPAK6 viral DNA (CLONTECH) and one of the mutated cGB-8/14 clones in the pBacPAK9 expression vector by the lipofection method according to the protocol from CLONTECH. At 3 days post-infection, the cotransfection supernatant was collected, amplified twice in Sf9 cells, and then used directly as virus stock for expression without additional purification of recombinant viruses. High Five cells (Invitrogen) grown at 27 °C in complete Grace's insect medium (Invitrogen) with 10% fetal bovine serum (Intergen) and 10 µg/ml gentamycin (Life Technologies, Inc.) in T-185 flasks were infected by 5 ml of virus stock/flask. The culture medium was harvested at 96 h post-infection. Recombinant enzyme production was calculated from Equation 1.
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(Eq. 1) |
Purification of Wild-type and Mutant cGB-PDEs--
The culture
medium (~250 ml) was fractionated by sequential ammonium sulfate
precipitation at 4 °C. The fraction precipitated by 25-40%
saturation was resuspended in 30 ml of 10 mM sodium phosphate buffer, pH 7.2, and centrifuged at 48,000 × g for 30 min at 4 °C. The supernatant was loaded onto a
hydroxyapatite (Bio-Rad) column (1.5 × 15 cm) equilibrated with
10 mM sodium phosphate buffer, pH 7.2. The column was
washed with 100 ml of 70 mM sodium phosphate buffer, pH
7.2, and then eluted with 120 mM sodium phosphate buffer,
pH 7.2, at a flow rate of 5 ml/h. The pool containing cGB-PDE activity
was diluted with six volumes of ice-cold deionized water and
concentrated to approximately 1 ml using an Amicon filtration cell
equipped with a PM-30 membrane. All purification steps were performed
at 4 °C. The final preparation was stored in 20% glycerol at
70 °C.
Catalytic Activity of cGB-PDE-- PDE activity was measured using a modification of the assay procedure described previously (12). Incubation mixtures contained 40 mM MOPS, pH 7.5, 0.5 mM EGTA, 15 mM magnesium acetate, 0.15 mg/ml bovine serum albumin, 20 µM cGMP (unless otherwise stated), [3H]cGMP (100,000-150,000 cpm/assay), and one of the cGB-PDE samples, in a total volume of 250 µl. The incubation time was 10 min at 30 °C. The reaction was stopped by placing the tubes in a boiling water bath for 3 min. After cooling, 20 µl of 10 mg/ml C. atrox snake venom was added, followed by a 20-min incubation at 30 °C. Nucleoside products were separated from unreacted nucleotides on the columns with DEAE Sephadex A-25 equilibrated with 20 mM Tris-HCl buffer, pH 7.5, and counted. In all studies, less than 15% of the total [3H]cGMP was hydrolyzed during the reaction. The apparent Km and Vmax values were determined from Lineweaver-Burk plots after assaying PDE activity in duplicate at 1-250 µM cGMP. kcat was obtained by dividing Vmax by the molar enzyme concentration. The molar enzyme concentration was calculated as described below under "Other Methods." To determine IC50 values for zaprinast, the PDE activity was assayed in duplicate in the presence of 0.5-30 µM zaprinast. All values determined represent at least three measurements using at least two different PDE preparations.
cGMP Saturation Binding-- The cGMP saturation binding assay was conducted in a total volume of 60 µl containing 10 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, 0.2 mM 3-isobutyl-1-methylxanthine, 0.5 mg/ml histone VIII-S, and 0.5-25 µM [3H]cGMP. The reaction was initiated by addition of an aliquot of enzyme. Following a 60-min incubation on ice, assay mixtures were filtered onto premoistened Millipore HAWP filters (pore size, 0.45 µm), which were then rinsed four times with a total of 4 ml of cold 10 mM sodium phosphate buffer, pH 6.8, with 1 mM EDTA, and then dried and counted.The data were corrected by subtraction of nonspecific binding, which was defined as either the [3H]cGMP bound in the absence of cGB-PDE or the [3H]cGMP bound in the presence of a 100-fold excess of unlabeled cGMP. A similar 2-4% of nonspecific binding was obtained with each method. The data were subjected to nonlinear least squares analysis using the program MINSQ II (Micromath Scientific Software, Salt Lake City, UT) to obtain the dissociation constant (Kd).
Other Methods-- SDS-electrophoresis in 10% polyacrylamide gels and Western blot analysis were done as described previously (12). Total protein concentrations were determined by the method of Bradford (14) using bovine serum albumin as the standard. To determine the cGB-PDE protein concentration, the Coomassie Brilliant Blue-stained SDS-polyacrylamide gels of wild-type and mutant enzymes were scanned using an E-C Apparatus Corp. densitometer equipped with GS370 v.3.0 software from Hoeffer. The cGB-PDE protein concentration was calculated from the fraction of the cGB-PDE band times the total protein concentration determined by Bradford assay. To convert the cGB-PDE protein concentration into the molar cGB-PDE concentration, the value of the molecular weight of cGB-PDE of 98.5 kDa (calculated from the amino acid sequence of cGB-PDE) was used.
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RESULTS |
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Mutagenesis Strategy-- The sequence alignments of the conserved catalytic domain of different PDEs have been published (2, 10, 15, 16). These studies revealed two blocks of conserved amino acid residues (Tyr596-His675 and Asp754-Glu783 in the case of cGB-PDE) separated by a variable sequence containing two invariant residues (Thr713 and Asp714 in the case of cGB-PDE) located approximately in the middle of this sequence (Fig. 1). It has been suggested that the first block is responsible for Zn2+ binding and could be part of the catalytic machinery of PDEs (13). Mutational studies on one of the PDE4 isozymes have shown that replacement of invariant His278, His311, or Thr349 (corresponding to His643, His675, or Thr713 in cGB-PDE) decreased the Vmax of this enzyme, but Km measurements for substrate were not reported (7). The second block possesses some general sequence similarity with the allosteric cGMP-binding sites (12) and could be involved in substrate binding. These findings prompted us to systematically assess the functional role of individual conserved amino acids using scanning mutagenesis.
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Expression and Purification of Wild-type and Mutated Forms of cGB-PDE-- Wild-type and mutants of the bovine lung cGB-PDE were expressed in High Five cells as described under "Experimental Procedures." The levels of expression of most of the mutants were comparable to that of the wild-type enzyme. The total production of recombinant cGB-PDEs was approximately 1-6 mg/100 ml of culture. The wild-type and mutant cGB-PDEs were partially purified similarly from culture medium using ammonium sulfate precipitation and hydroxyapatite chromatography as described under "Experimental Procedures." There was no noticeable difference in binding to and subsequent elution of these proteins from the hydroxyapatite column compared with that for the wild-type enzyme. Fig. 2 shows a Coomassie Blue-stained SDS-polyacrylamide gel of partially purified mutants obtained following the hydroxyapatite column step. All mutated cGB-PDEs migrated with essentially the same mobility as that of the wild-type enzyme. The identity of the recombinant proteins was verified by Western blot analysis (data not shown).
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Kinetic Analysis of Mutants-- The kinetic parameters, Km for cGMP and kcat (Table I), were determined from Lineweaver-Burk plots. The contribution of the substituted amino acid side chain to binding energy in enzyme-transition state complexes was calculated from values of the catalytic efficiency (kcat/Km) using Equation 2.
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(Eq. 2) |
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Mutants Defective in kcat-- Nine mutants have kcat that is less than 15% of the wild-type value (Fig. 3), including two mutants (H643A and D754A) that retain only 0.4% of wild-type kcat. Substitution within the second block of conserved amino acid residues (Fig. 1) had little effect on kcat value, except for substitution of the invariant Asp754. Mutations with markedly decreased kcat were primarily clustered around the conserved HX3HXnE motifs of the putative Zn2+-binding site (13). The mutation of the Asp714 in the invariant TD dyad also displayed significantly reduced PDE activity.
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Mutants Defective in kcat and Km-- H647A and E672A mutants possess a 10- and 14-fold increase in Km for cGMP, and an 8- and 13-fold decrease in kcat, respectively. The role of these residues cannot be interpreted unambiguously. They may be involved in catalysis, important for recognition of substrate, or provide a structural role.
Mutants Defective in Km--
Four mutants (Y602A,
T713A, E775A, and Q779A) were defective mainly in Km
(Table I). Two of these (Thr713 and Gln779) are
uncharged amino acids and, despite the moderate changes in
Km when these are substituted with alanine, the
GT for each of these mutants was in the range
that is predicted for loss of a hydrogen bond between an enzyme polar
side chain and the substrate (19). Two mutants, Y602A and E775A,
exhibited profound losses in affinity for cGMP with
Km values of 65 and 70 µM,
respectively, compared with a Km of 2 µM for wild-type cGB-PDE. The
GT
for each of these mutants is 2.9 or 2.8 kcal/mol, respectively, which
is within the range expected for the loss of a salt bridge
(electrostatic interaction) (18) and approaching the range (3.5-4.5
kcal/mol) expected for the loss of a hydrogen bond involving a charged
residue (19).
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Zaprinast-binding Site-- Zaprinast selectively inhibits cGMP-specific PDEs (20). Fig. 4 shows that double reciprocal plots for wild-type cGB-PDE at various concentrations of zaprinast intersect at 1/Vmax; this indicates a competitive inhibitory mechanism for zaprinast. By determining the values of Km for cGMP at different concentrations of zaprinast, the value for Ki was 0.15 µM under the experimental conditions used. As a competitive inhibitor, zaprinast should directly compete with cGMP for the active site of cGB-PDE. Thus, zaprinast may be used to probe the structure of the cGB-PDE active site. IC50 values for zaprinast using the wild-type and all of the mutant enzymes are shown in Table I. To generate each IC50 value, the cGMP concentration in the assay was one-third the Km for each mutant tested. When using low substrate concentrations, IC50 values approach the Ki for a competitive inhibitor. To decipher how the replacement of conserved residues in the catalytic domain of cGB-PDE affects the affinity for zaprinast in comparison with cGMP, the IC50 for zaprinast and the Km for cGMP of wild-type enzyme were taken as 1.0 and the corresponding values for mutants were calculated as a fold change for each parameter. Fig. 5 shows that many mutants (Y596A, H603A, N604A, H607A, E632A, D644A, H675A, D714A, S756A, K760M, F776L, D781A, and E783A) with small changes in Km for cGMP had small changes in IC50 for zaprinast, suggesting that residues replaced in these mutants do not participate directly either in cGMP binding or in interaction with zaprinast. Some mutants (for example, H643A or H647A) had moderate changes in the both parameters. It is possible that residues replaced in these mutants are important for catalysis or for structural integrity of the catalytic site rather than for direct interaction with cGMP or zaprinast. The fact that the largest changes in Km (Y602A, E672A, T713A, E775A, and Q779A) did not correlate with the largest changes in IC50 (D754A and G780A) was unexpected. The greatest increase in the IC50 value was found for the D754A mutant, which also exhibited a profound change in catalytic activity. This residue could be a major target of zaprinast action, but the selectivity of zaprinast inhibition of the cGMP-specific PDEs must be provided by other components of the catalytic domain since aspartic acid in this position is conserved in all mammalian PDEs.
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Structural Integrity of Mutants-- To address structural integrity of the mutant enzymes, the deviation of all three kinetic parameters (Km, kcat, and IC50) for each mutant from the same parameters of wild-type enzyme was calculated. If the value for any mutant exhibited a small change (7-fold or less), this would imply that this mutant preserved the overall structure typical for wild-type enzyme, and this mutant was not included for further characterization. By following this rule, Y602A, H643A, H647A, E672A, D754A, E775A, and E775Q mutants were selected for additional characterization. cGB-PDE contains two allosteric cGMP-binding sites that are located toward the NH2 terminus of the protein molecule, and which are distinct from the site of cGMP hydrolysis. The [3H]cGMP filter-binding assay was used to examine the affinity of cGMP binding to these sites. The [3H]cGMP-binding concentration curves were almost indistinguishable (data not shown) for these proteins, and the data subjected to nonlinear least squares analysis did not show a significant difference in dissociation constant (Kd). The Kd values of wild-type and Y602A, H643A, H647A, E672A, D754A, E775A, and E775Q mutants were found to be approximately equal (1.3, 1.8, 1.3, 1.3, 1.5, 1.3, 2.0, and 1.8 µM, respectively). The binding stoichiometry for these seven mutants was in the range 0.5-0.6 mol of [3H]cGMP/monomer, which approximates the value for native cGB-PDE reported previously (6).
Taken together, these data imply that differences in Km, kcat, and IC50 values (Tables I and II) are not due to nonspecific conformational effects induced by the mutations, and that all mutants preserved the overall structure typical for wild-type enzyme. ![]() |
DISCUSSION |
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The strategy of scanning mutagenesis is widely used to identify potentially important residues involved in protein function. Once identified, these residues can be analyzed more extensively to further examine their functional and structural role. In this study, the possible role of 23 amino acid residues that are conserved in all known catalytic domain of PDEs has been evaluated by systematic substitution of these residues in cGB-PDE. All mutants were expressed as full-length enzymes with similar levels of production and the same chromatographic properties. This indicates that the substitutions did not cause gross perturbations to the tertiary conformation and subsequent destabilization or proteolysis of the enzymes.
An interesting finding was that the residues that are most important for catalysis (His643 and Asp754) and for substrate binding (Tyr602 and Glu775) are not located in two different blocks of conserved residues. Furthermore, the invariant TD dyad, which is located in the highly variable sequence between these two blocks, contributes to catalysis (Asp714) and substrate binding (Thr713). Such distribution of the functionally important residues assumes that the active site may be formed at the interface between these blocks of conserved residues. Thus, the catalytic and substrate-binding components are overlapping.
Nine residues (His603, Asn604, His607, His643, Asp644, His647, Glu672, Asp714, and Asp754) have been found to be important for catalysis in cGB-PDE. Substitution of either of two of them, His643 or Asp754, is accompanied by the largest decrease in catalytic efficiency. His643 of cGB-PDE corresponds to His278 of a cAMP-specific PDE (RNPDE4D). The latter residue was shown to be critical for PDE4 activity (7), and our results are consistent with this observation. In addition to His278, two more residues (His311 and Thr349) of PDE4 were found to be critical for catalytic activity in PDE4 (7). Substitution of the corresponding positions of cGB-PDE (His675 and Thr713) produced little effect on catalysis. Instead of a possible His/Thr catalytic dyad in PDE4, a possible His/Asp catalytic dyad was uncovered in cGB-PDE (PDE5). This could be an interesting point of discrimination between cAMP-specific PDEs and cGMP-specific PDEs.
A full understanding of PDE catalysis is complicated by the fact that the activities of PDEs are supported by a number of divalent metal cations (13, 21, 22). The catalytic activity of cGB-PDE is supported by Zn2+, Mn2+, Co2+, and Mg2+, but Zn2+ promotes cGB-PDE activity at lower concentrations than do other cations (13). Sequence alignment reveals two putative Zn2+-binding motifs which are conserved in all mammalian PDEs (13). These motifs include His603, His607, and Glu632 followed by His643, His647, and Glu672 residues of cGB-PDE. All of these residues were replaced with alanine in the present study, and five of six of these mutants exhibited a marked decrease in catalytic efficiency (Table I). The only residue in this group which seems nonessential for catalysis is Glu632. When histidine residues of HSPDE4A at positions 433, 437, 473, and 477, which correspond to His603, His607, His643, and His647 of cGB-PDE, respectively, were changed independently to serine residues, cAMP hydrolyzing activity of HSPDE4A was substantially reduced (23). Our results are consistent with this observation. Asn604 and Asp644 are flanking residues for His603 and His643, respectively. In traditional Zn2+-binding sites of different hydrolases, the flanking position of the first histidine is occupied by glutamic acid (His-Glu-X2-His-Xn-Glu). Mutagenesis of this glutamic acid led to a drastic decrease of the catalytic activity of aminopeptidase A (24). Replacement of Asn604 or Asp644 by alanine in cGB-PDE decreased the kcat approximately 9-fold. Unfortunately, the basal activity of cGB-PDE in the metal-free assay was strongly diminished, and for mutants defective in kcat (in the range of 10-250-fold) it was technically difficult to measure the enhancement of activity due to Zn2+. Nevertheless, our data emphasize an important role for His603, His607, His643, His647, Glu672, Asp714, and Asp754 for catalysis, and one possible function of this combination of residues is to provide a Zn2+-binding site(s). However, it appears that a single Zn2+-binding motif is insufficient to support normal catalysis in PDEs. It should also be mentioned that a His/His/Glu site has been described for binding Mn2+ in the active site of 3,4-dihydroxyphenylacetate 2,3-dioxygenase (25), and several enzymes contain multinuclear metal-binding sites that are created by multiple histidines and acidic residues. The PDE2 exhibits highest activity with Mn2+ (21), and cGB-PDE activity is also supported by Mn2+ (13). It is possible that the same site of cGB-PDE, which tightly binds Zn2+, can bind other divalent cations as well. However, in studies of the rod outer segment PDE (PDE6), prolonged treatment with chelators inactivates the enzyme even in the presence of Mn2+, Mg2+ or Co2+, and catalytic activity is restored by addition of Zn2+.2
Y602A, T713A, E775A, and Q779A mutants were defective in
Km with small or negligible changes in
kcat. The kcat values of
these mutants are critical for interpretation, because it is assumed
that Tyr602, Thr713, Glu775, and
Gln779 residues of cGB-PDE are important for binding the
cGMP substrate. Unfortunately, the magnitude of Km
changes for T713A and Q779A mutants cannot be interpreted
unambiguously, because the values for the calculated
GT are maximum values that include any loss of
binding energy due to small perturbations of the overall conformation
of the enzyme. Only the amino acid positions whereby substitutions
cause large loss of function can be considered essential.
Alternatively, the residues whose substitution lead to moderate changes
in substrate binding may be involved in the general arrangement of the
substrate-binding site, and do not necessarily interact directly with
substrate. For this reason, only two residues (Tyr602 and
Glu775), for which substitution exhibited the largest
changes in Km (Table I) were selected for further
analysis, and additional mutations (Y602F, E775D, and E775Q) were
generated and analyzed (Table II). The possible roles of
Tyr602 and Glu775 in cGMP binding in the
catalytic site of cGB-PDE are discussed below.
Tyrosine 602-- There are several options for the tyrosine residue to interact with cGMP: 1) a hydrogen bond to an oxygen atom on the phosphoryl group, 2) a hydrogen bond to the 2'-OH group of the ribose ring, and 3) stacking interaction with the base. These types of interactions for a tyrosine residue have been found in the complex of ribonuclease T1 with 2'-GMP (26), in the nonphysiological complex of nucleoside diphosphate kinase with cAMP (27), in the AMP-binding site of fructose-1,6-bisphosphatase (28), and in site B of the regulatory subunit of protein kinase A (29). To scrutinize the possible functions of Tyr602 in the cGB-PDE catalytic site, results of cyclic nucleotide analogs as competitive inhibitors of cGB-PDE (30) can also be considered. The published results indicate that the 2'-OH group of cGMP is not a major requirement for binding to the catalytic site of cGB-PDE (30, 31). Tyr602 is invariant in all mammalian PDEs, but in Drosophila dunce PDE (32) and in Saccharomyces cerevisiae PDE (33), the corresponding Tyr602 position is occupied by Phe. Phe could mimic Tyr for possible participation in stacking interactions with the guanine base. The Y602F mutant was indistinguishable from wild-type enzyme in terms of Km and kcat values (Table II). This is a strong argument that Tyr602 stacks with the purine base of cGMP in the substrate-binding site of cGB-PDE and is likely to serve a similar role in other PDEs.
Glutamic Acid 775-- There are two major chemical options for the glutamic acid residue to interact with cGMP: 1) a hydrogen bond interaction with the 2'-OH group of the ribose ring, such as that found in the catabolite gene activator protein (34) or in the regulatory subunit of protein kinase A (29), and 2) simultaneous interaction with the C-2 exocyclic amino group and the hydrogen atom of the N-1 endo-nitrogen of the guanine base, such as that found in the ribonuclease T1 (26). It has been mentioned above that the 2'-OH group on the ribose ring appears to be unimportant for cGMP binding to the PDE catalytic site (30, 31). In addition, cGMP analog studies suggest that the N-1 endo-nitrogen of cGMP contributes to binding at the catalytic site, whereas the 2-amino group of cGMP is not a major requirement for interaction (30, 31). Thus, a possible function of Glu775 could be for interaction with the hydrogen atom of the N-1 endo-nitrogen of the guanine base. The fact that the E775D mutant exhibits only small changes in Km compared to the E775A or E775Q mutants confirmed that an acidic residue in this position is important for cGMP binding.
Recently, some of the major interactions for cGMP in the allosteric binding site a of cGB-PDE have been identified (11). Comparison with the present results for the catalytic site reveals little similarity between cGMP binding in the catalytic and allosteric sites of the cGB-PDE, except for the fact that, in both sites, a negatively charged residue (Glu in the catalytic site and Asp in the allosteric site) is involved in interactions with cGMP. The substrate cyclic nucleotide-binding site of PDEs could therefore represent a newly recognized third group of mammalian cyclic nucleotide receptors. The first group includes the cyclic nucleotide-dependent protein kinases and the cyclic nucleotide-gated cation channels (35); the second group includes allosteric cGMP-binding sites of PDEs (11). PDEs are important targets for therapeutic drugs, and it is now feasible to use the data of site-directed mutagenesis as a basis for drug design. This involves definition of the residues responsible for the interactions between the substrate or drug and the protein. It is interesting that zaprinast is a competitive inhibitor and should bind to the cGMP-binding site; however, the major contact points for these two compounds are quite different. This could explain in part the inhibitory effect of zaprinast, which might maintain a different orientation in the binding site, and could interact with a residue which is critical for catalysis (such as Asp754). In summary, we have assessed the effect of substitution at 23 positions in the catalytic domain of the cGB-PDE which are conserved in all known mammalian PDEs. To our knowledge, this is the most comprehensive mutational analysis of any PDE reported to date. The major observations of this study are as follows: (i) His643 and Asp754 are critical for cGB-PDE catalytic activity; (ii) His603, His607, His643, His647, Glu672, Asp714, and Asp754 may be part of a metal-binding site which is important for catalysis; (iii) Tyr602 and Glu775 are critical for cGMP binding, and Tyr602 is probably involved in stacking interactions with the guanine base of cGMP; (iv) the zaprinast-binding site is overlapping with the substrate cGMP-binding site, but some of the residues for binding these two ligands differ. ![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM41269.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 615-322-4384;
Fax: 615-343-3794; E-mail: jackie.corbin{at}mcmail.vanderbilt.edu.
1 The abbreviations used are: PDE, 3':5'-cyclic nucleotide phosphodiesterase; cGB-PDE, cGMP-binding cGMP-specific phosphodiesterase; MOPS, 3-(N-morpholino)propanesulfonic acid.
2 S. H. Francis, unpublished results.
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