Portal de Periódicos da CAPES

Potential Roles of Conserved Amino Acids in the Catalytic Domain of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

1998; Elsevier BV; Volume: 273; Issue: 11 Linguagem: Inglês

10.1074/jbc.273.11.6460

1083-351X

Illarion V. Turko, Sharron H. Francis, Jackie D. Corbin,

Receptor Mechanisms and Signaling

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, k cat, and IC50 for zaprinast. The most dramatic decrease in k cat was seen with H643A and D754A mutants with the decrease in free energy of binding (ΔΔGT) 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 k cat, 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 ΔΔGT 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. 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, k cat, and IC50 for zaprinast. The most dramatic decrease in k cat was seen with H643A and D754A mutants with the decrease in free energy of binding (ΔΔGT) 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 k cat, 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 ΔΔGT 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. The 3′:5′-cyclic nucleotide phosphodiesterase (PDE) 1The abbreviations used are: PDE, 3′:5′-cyclic nucleotide phosphodiesterase; cGB-PDE, cGMP-binding cGMP-specific phosphodiesterase; MOPS, 3-(N-morpholino)propanesulfonic acid. 1The abbreviations used are: PDE, 3′:5′-cyclic nucleotide phosphodiesterase; cGB-PDE, cGMP-binding cGMP-specific phosphodiesterase; MOPS, 3-(N-morpholino)propanesulfonic acid. 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 (1Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar). Comparison of the reported PDE sequences reveals a conserved region of approximately 270 amino acids located toward the COOH terminus of PDE molecules (2Charbonneau H. Beier N. Walsh K.A. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9308-9312Crossref PubMed Scopus (111) Google Scholar). 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 (3Tucker M.M. Robinson Jr., J.B. Stellwagen E. J. Biol. Chem. 1981; 256: 9051-9058Abstract Full Text PDF PubMed Google Scholar, 4Kincaid R.L. Stith-Coleman I.E. Vaughan M. J. Biol. Chem. 1985; 260: 9009-9015Abstract Full Text PDF PubMed Google Scholar, 5Stroop S.D. Charbonneau H. Beavo J.A. J. Biol. Chem. 1989; 264: 13718-13725Abstract Full Text PDF PubMed Google Scholar, 6Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14964-14970Abstract Full Text PDF PubMed Google Scholar), deletion mutagenesis (7Jin S.-L.C. Swinnen J.V. Conti M. J. Biol. Chem. 1992; 267: 18929-18939Abstract Full Text PDF PubMed Google Scholar, 8Cheung P.P. Xu H. McLaughlin M.M. Ghazaleh F.A. Livi G.P. Colman R.W. Blood. 1996; 88: 1321-1329Crossref PubMed Google Scholar, 9Jacobitz S. McLaughlin M.M. Livi G.P. Burman M. Torphy T.J. Mol. Pharmacol. 1996; 50: 891-899PubMed Google Scholar), and point mutations targeting conserved residues (7Jin S.-L.C. Swinnen J.V. Conti M. J. Biol. Chem. 1992; 267: 18929-18939Abstract Full Text PDF PubMed Google Scholar) 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 (10McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Le Trong H. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar). 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 (11Turko I.V. Haik T.L. McAllister-Lucas L.M. Burns F. Francis S.H. Corbin J.D. J. Biol. Chem. 1996; 271: 22240-22244Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) 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 (10McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Le Trong H. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar). One of these blocks has some sequence similarity to the allosteric binding sites (12McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Crossref PubMed Scopus (83) Google Scholar), 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 (13Francis S.H. Colbran J.L. McAllister-Lucas L.M. Corbin J.D. J. Biol. Chem. 1994; 269: 22477-22480Abstract Full Text PDF PubMed Google Scholar). 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, k cat, and IC50 for zaprinast, respectively. [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. cGB-8/14 clone encodes a full-length bovine lung cGB-PDE (11Turko I.V. Haik T.L. McAllister-Lucas L.M. Burns F. Francis S.H. Corbin J.D. J. Biol. Chem. 1996; 271: 22240-22244Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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 AGCGCT 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 GATGCC 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 CATGCT 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 CATGCT 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 GCCGCA 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-X™ 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. 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. cGB­PDE in sample=(total protein in sample)(specific enzyme activity in sample)(n)(specific enzyme activity of purified cGB­PDE)Equation 1 Specific enzyme activity for purified cGB-PDE was taken as 2.5 μmol/mg/min, and n is defined ask cat,wild-type/k cat,mutant. 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. PDE activity was measured using a modification of the assay procedure described previously (12McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Crossref PubMed Scopus (83) Google Scholar). 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 apparentKm and V max values were determined from Lineweaver-Burk plots after assaying PDE activity in duplicate at 1–250 μm cGMP. k catwas obtained by dividing V max 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. The cGMP saturation binding assay was conducted in a total volume of 60 μl containing 10 mmsodium 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). SDS-electrophoresis in 10% polyacrylamide gels and Western blot analysis were done as described previously (12McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Crossref PubMed Scopus (83) Google Scholar). Total protein concentrations were determined by the method of Bradford (14Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar) 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. The sequence alignments of the conserved catalytic domain of different PDEs have been published (2Charbonneau H. Beier N. Walsh K.A. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9308-9312Crossref PubMed Scopus (111) Google Scholar, 10McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Le Trong H. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar, 15Swinnen J.V. Joseph D.R. Conti M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5325-5329Crossref PubMed Scopus (143) Google Scholar, 16Meacci E. Taira M. Moos Jr., M. Smith C.J. Movsesian M.A. Degerman E. Belfrage P. Manganiello V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3721-3725Crossref PubMed Scopus (144) Google Scholar). 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 (13Francis S.H. Colbran J.L. McAllister-Lucas L.M. Corbin J.D. J. Biol. Chem. 1994; 269: 22477-22480Abstract Full Text PDF PubMed Google Scholar). 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 V max of this enzyme, but Kmmeasurements for substrate were not reported (7Jin S.-L.C. Swinnen J.V. Conti M. J. Biol. Chem. 1992; 267: 18929-18939Abstract Full Text PDF PubMed Google Scholar). The second block possesses some general sequence similarity with the allosteric cGMP-binding sites (12McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Crossref PubMed Scopus (83) Google Scholar) 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. Twelve amino acid residues in the first block were substituted singly by alanine. Thr and Asp of the TD dyad and seven residues in the second block were also replaced by alanine. Lys and Phe in the second block were replaced by Met and Leu, respectively. 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). The kinetic parameters, Km for cGMP and k cat (TableI), 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 (k cat/Km) using Equation2. ΔΔGT=−RTln[(kcat/Km)mutant/(kcat/Km)wild­type]Equation 2 ΔΔGT is the change in the free energy of binding in enzyme-transition state complexes attributable to the substituted group (17Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (254) Google Scholar). R, the ideal gas constant, is equal to 1.98 × 10−3 kcal/degree/mol, and T, the temperature at which the assay was done, is equal to 303 K. The effect of substitution of the amino acid side chain that interacts with a substrate may be manifested in terms of k cat, Km, or both (17Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (254) Google Scholar). In the present study, the binding of substrate in the transition state was chosen because the intrinsic binding energy of groups on the enzyme and substrate may not be fully realized until the enzyme-transition state complex is formed, whereby some of the binding energy may be diverted to stabilize the transition state.Table IKinetic parameters of the catalytic domain mutants of cGB-PDEcGB-PDEKmk catk cat/Km(× 106)ΔΔG TIC50 for zaprinastμms−1M−1s−1kcal/molμmWild-type2 ± 0.44.27 ± 0.132.140.3 ± 0.01Y596A6 ± 1.02.34 ± 0.070.391.00.8 ± 0.02Y602A65 ± 5.01.12 ± 0.030.0172.92.0 ± 0.06H603A6 ± 0.90.11 ± 0.0030.0182.91.7 ± 0.05N604A6 ± 0.90.48 ± 0.010.082.00.8 ± 0.02H607A5 ± 0.90.12 ± 0.0040.0242.71.0 ± 0.03E632A5 ± 0.91.60 ± 0.050.321.10.5 ± 0.02H643A14 ± 2.60.016 ± 0.00050.0014.52.4 ± 0.07D644A7 ± 1.00.53 ± 0.020.0762.01.1 ± 0.03H647A20 ± 2.80.55 ± 0.020.0282.63.0 ± 0.09E672A28 ± 3.00.33 ± 0.010.0123.10.4 ± 0.01H674A12 ± 2.02.30 ± 0.070.191.54.1 ± 0.12H675A10 ± 2.03.46 ± 0.10.351.10.3 ± 0.01T713A30 ± 3.03.15 ± 0.090.1051.80.3 ± 0.01D714A5 ± 0.90.096 ± 0.0030.0192.81.0 ± 0.03D754A15 ± 2.60.015 ± 0.00050.0014.613.0 ± 0.4S756A3 ± 0.42.10 ± 0.060.700.70.5 ± 0.02K760M3 ± 0.42.12 ± 0.060.710.70.8 ± 0.02E775A70 ± 5.01.53 ± 0.050.0222.81.3 ± 0.04F776L6 ± 0.92.04 ± 0.060.341.10.7 ± 0.02Q779A16 ± 2.64.18 ± 0.130.261.31.3 ± 0.04G780A9 ± 1.53.73 ± 0.110.411.010.0 ± 0.3D781A8 ± 1.51.33 ± 0.040.171.50.8 ± 0.02E783A12 ± 2.02.56 ± 0.080.211.41.5 ± 0.05 Open table in a new tab Previous studies have determined the magnitude of the changes in transition state binding expected for the disruption of particular interactions between enzymes and substrates (18Wells J.A. Powers D.B. Bott R.R. Graycar T.P. Estell D.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1219-1223Crossref PubMed Scopus (214) Google Scholar, 19Fersht A.R. Shi J.-P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Carter P. Waye M.M.Y. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (986) Google Scholar). Deletion of a charged group to disrupt a hydrogen bond between the enzyme and a substrate weakened the binding energy by 3.5–4.5 kcal/mol (19Fersht A.R. Shi J.-P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Carter P. Waye M.M.Y. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (986) Google Scholar), whereas the disruption of an electrostatic interaction between a charged group in the enzyme and substrate weakened binding by 2.0 kcal/mol (18Wells J.A. Powers D.B. Bott R.R. Graycar T.P. Estell D.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1219-1223Crossref PubMed Scopus (214) Google Scholar). It is important to emphasize that the values for the calculated ΔΔGT are maximum values that include any loss of binding energy due to small perturbations of the overall conformation of the enzyme. Therefore, 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 may be involved in the general arrangement of the catalytic site. Based on calculated ΔΔGT (Table I), the mutants could be arbitrarily placed into two groups. The first group includes Y596A, E632A, H674A, H675A, S756A, K760M, F776L, G780A, D781A, and E783A mutants. The changes found for these mutants are not sufficient to suggest an essential role for these residues. The second group of 13 mutants have ΔΔGT values in the range expected for important roles for these amino acid residues in the wild-type enzyme. This group could be divided into three categories: those defective mainly in k cat, those defective mainly in Km, and those defective in both of these parameters. Nine mutants havek cat 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-typek cat. Substitution within the second block of conserved amino acid residues (Fig. 1) had little effect onk cat value, except for substitution of the invariant Asp754. Mutations with markedly decreasedk cat were primarily clustered around the conserved HX 3HXnE motifs of the putative Zn2+-binding site (13Francis S.H. Colbran J.L. McAllister-Lucas L.M. Corbin J.D. J. Biol. Chem. 1994; 269: 22477-22480Abstract Full Text PDF PubMed Google Scholar). The mutation of the Asp714 in the invariant TD dyad also displayed significantly reduced PDE activity. Five mutants (H603A, N604A, H607A, D644A, and D714A) were defective in k cat only (Table I). These residues may be directly involved in catalysis, or they may provide important structural features that allow for effective catalysis. Substitution of these residues may perturb the configuration of the active site. H643A and D754A mutants have a 7-fold increase in Km; however, this defect is insignificant in comparison to the large decrease (270- and 280-fold, respectively) in k cat. One possible interpretation of such large changes by mutation of His643 and Asp754 is that these residues represent a catalytic dyad. H647A and E672A mutants possess a 10- and 14-fold increase in Km for cGMP, and an 8- and 13-fold decrease in k cat, 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. 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 ΔΔGT 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 (19Fersht A.R. Shi J.-P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Carter P. Waye M.M.Y. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (986) Google Scholar). 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 ΔΔGTfor 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) (18Wells J.A. Powers D.B. Bott R.R. Graycar T.P. Estell D.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1219-1223Crossref PubMed Scopus (214) Google Scholar) and approaching the range (3.5–4.5 kcal/mol) expected for the loss of a hydrogen bond involving a charged residue (19Fersht A.R. Shi J.-P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Carter P. Waye M.M.Y. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (986) Google Scholar). To further probe the possible function of Tyr602 and Glu775 in cGMP binding to the substrate site, three additional mutants (Y602F, E775D, and E775Q) were generated, expressed and partially purified using the experimental procedures described for the major set of mutants. Y602F, E775D, and E775Q possessed the same level of expression, the same chromatographic behavior on hydroxyapatite columns, and exhibited the same mobility on the SDS-polyacrylamide gel as did wild-type enzyme. The identity of these mutants was verified by Western blot analysis (data not shown). Kinetic parameters of the Y602F mutant were indistinguishable from