“Catch 222,” the Effects of Symmetry on Ligand Binding and Catalysis in R67 Dihydrofolate Reductase as Determined by Mutations at Tyr-69
2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês
10.1074/jbc.m404485200
ISSN1083-351X
AutoresLori G. Stinnett, R. Derike Smiley, Stephanie N. Hicks, Elizabeth E. Howell,
Tópico(s)RNA and protein synthesis mechanisms
ResumoR67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in Km values for both ligands and a 2-fold rise in the kcat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344–11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the Kd and Km values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type Km value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate. R67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in Km values for both ligands and a 2-fold rise in the kcat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344–11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the Kd and Km values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type Km value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate. Dihydrofolate reductase (DHFR) 1The abbreviations used are: DHFR, dihydrofolate reductase; DHF, dihydrofolate; NMNH, reduced nicotinamide mononucleotide; NADP(+/H), oxidized/reduced nicotinamide adenine dinucleotide phosphate; pABA-glutamic acid tail, para-aminobenzoylglutamic acid region of dihydrofolate/folate; ITC, isothermal titration calorimetry; wt, wild-type; Quad3, the protein product of a tandem array of four inframe R67 DHFR genes; MES, 4-morpholineethanesulfonic acid. catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. DHFR activity is necessary for cell survival as tetrahydrofolate is involved in pathways leading to the synthesis of purine nucleosides and other metabolites (1Kraut J. Matthews D.A. Jurnak F.A. McPherson A. Biological Macromolecules & Assemblies. 3. John Wiley & Sons, New York1987: 1-71Google Scholar). Chromosomal (Escherichia coli) DHFR is inhibited by the antibiotic trimethoprim. However, plasmid-encoded R67 DHFR provides resistance to the antibiotic. The plasmid-encoded DHFR is unique in that it shows no genetic or structural homologies with chromosomal DHFR (2Narayana N. Matthews D.A. Howell E.E. Nguyen-huu X. Nat. Struct. Biol. 1995; 2: 1018-1025Crossref PubMed Scopus (76) Google Scholar, 3Matthews D.A. Smith S.L. Baccanari D.P. Burchall J.J. Oatley S.J. Kraut J. Biochemistry. 1986; 25: 4194-4204Crossref PubMed Scopus (60) Google Scholar, 4Stone D. Smith S.L. J. Biol. Chem. 1979; 254: 10857-10861Abstract Full Text PDF PubMed Google Scholar). Several features of R67 dihydrofolate reductase are unusual. First, as a homotetramer with a monomer length of 78 amino acids, it is one of the smallest enzymes known to self-assemble into an active quaternary structure. Second, the structure possesses 222 symmetry as shown in Fig. 1 2The monomers of R67 DHFR are labeled ABDC going in a clockwise orientation in the crystal structure (1VIE and 1VIF in the Protein Data Bank). The residues in monomer A are labeled 1–78, whereas those in monomers B, C, and D are designated 101–178, 201–278, and 301–378, respectively. All four symmetry-related residues are implied when one residue of the homotetramer is described. The corresponding domains in Quad3 are relabeled 1234 (=ABCD) to minimize any confusion and provide consistent nomenclature. (2Narayana N. Matthews D.A. Howell E.E. Nguyen-huu X. Nat. Struct. Biol. 1995; 2: 1018-1025Crossref PubMed Scopus (76) Google Scholar). Third, a pore, 25 Å long, extends through the middle of the enzyme (like a doughnut hole). Fourth, the symmetry, coupled with use of a single active site pore, results in overlapping binding sites for the two different ligands used in the reaction. For example, R67 DHFR binds a total of two molecules, either two NADPH, two folate/DHF, or one NADPH plus one folate/DHF (5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholar). The first two are dead-end complexes, whereas the third is productive. (Interligand cooperativity patterns funnel the enzyme toward the productive ternary complex.) Fifth, site-directed mutagenesis results in four mutations per active site pore and large effects on binding and catalysis. Thus, it is difficult to produce local effects that could allow dissection of how each residue interacts with the ligands as well as the transition state. Although a generalized description of R67 DHFR catalysis exists (5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholar, 6Li D. Levy L.A. Gabel S.A. Lebetkin M.S. DeRose E.F. Wall M.J. Howell E.E. London R.E. Biochemistry. 2001; 40: 4242-4252Crossref PubMed Scopus (44) Google Scholar, 7Hicks S.N. Smiley R.D. Hamilton J.B. Howell E.E. Biochemistry. 2003; 42: 10569-10578Crossref PubMed Scopus (26) Google Scholar, 8Park H. Bradrick T.D. Howell E.E. Protein Eng. 1997; 10: 1415-1424Crossref PubMed Scopus (29) Google Scholar, 9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar, 10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar), additional detail can be obtained by introduction of asymmetry. To be able to introduce asymmetry in R67 DHFR, we have constructed a tandem gene array that allows control of the number and location of the mutation(s). Briefly, the tandem gene array contains four in-frame copies of the gene encoding wild-type (wt) R67 DHFR. Transcription and translation yield a monomeric protein (named “Quad3”) mimicking the wild-type enzyme. Quad3 is almost fully active (1.8-fold decrease in kcat/Km values), and all physical, binding, and steady-state kinetic studies indicate excellent agreement with wt R67 DHFR behavior (10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar, 11Bradrick T.D. Shattuck C. Strader M.B. Wicker C. Eisenstein E. Howell E.E. J. Biol. Chem. 1996; 271: 28031-28037Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Those residues identified as most important in R67 DHFR catalysis include Lys-32, Gln-67, Ile-68, and Tyr-69 (7Hicks S.N. Smiley R.D. Hamilton J.B. Howell E.E. Biochemistry. 2003; 42: 10569-10578Crossref PubMed Scopus (26) Google Scholar, 9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar, 12Strader M.B. Chopra S. Jackson M. Smiley R.D. Stinnett L.G. Wu J. Howell E.E. Biochemistry. 2004; 43: 7403-7412Crossref PubMed Scopus (19) Google Scholar). The first asymmetric mutation series constructed to probe binding and catalysis involved the Q67H substitution (10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar). The companion paper (Hicks et al. (42Hicks S.N. Smiley R.D. Stinnett L.G. Minor K.H. Howell E.E. J. Biol. Chem. 2004; 279: 46995-47002Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar)) describes construction and characterization of a series of K32M asymmetric mutants. This study focuses on the role of Tyr-69 residues. What is the proposed role for Tyr-69 in R67 DHFR function? Docking studies suggest that these symmetry-related residues may be involved in interactions with both the substrate and cofactor (13Howell E.E. Shukla U. Hicks S.N. Smiley R.D. Kuhn L.A. Zavodszky M.I. J. Comput. Aided Mol. Des. 2001; 15: 1035-1052Crossref PubMed Scopus (20) Google Scholar). The ring edges of two symmetry-related tyrosine 69 residues dock within van der Waals contact with the pyrophosphate bridge of NADPH as well as with the adenine ribose. Docking studies also predict a possible interaction between the tyrosine hydroxyl and the glutamic acid tail of folate. Kinetic studies of the homotetrameric Y69F mutant also support the hypothesis that Tyr-69 interacts with both DHF and NADPH, because the Km values for DHF and NADPH are 11- and 17-fold weaker, respectively, than those for wild-type R67 DHFR (9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar). In addition, the kcat for the reaction is 2-fold greater than the kcat for wild-type R67 DHFR. A less conservative substitution (Y69H) greatly increased both Km values as well as decreased kcat. Finally, a recent NMR study finds chemical shifts are associated with Tyr-69 upon NADP+ binding (14Pitcher 3rd, W.H. DeRose E.F. Mueller G.A. Howell E.E. London R.E. Biochemistry. 2003; 42: 11150-11160Crossref PubMed Scopus (19) Google Scholar). A model of the docked R67 DHFR·NADPH·folate ternary complex is shown in Fig. 1E with the positions of the symmetry-related Tyr-69 residues indicated. Our approach to understanding the role of symmetry-related tyrosine 69 residues in ligand binding and catalysis in this report is 2-fold. First, we generated a series of asymmetric Y69F mutants, including a single mutant (containing one Y69F mutation), three double mutants (each containing two Y69F mutations), a triple mutant, and a quadruple mutant to better understand the specificity of the interactions between Tyr-69 and both DHF and NADPH. This approach allows us to determine if there is a preference for NADPH and/or DHF to interact with wild-type tyrosine 69 residues. Our second approach involved site-directed mutagenesis of tyrosine 69 residues within the homotetrameric enzyme to better understand the type(s) of interaction(s) occurring between symmetry-related tyrosine 69 and the ligands and how these interactions are involved in catalysis. Construction of Asymmetric Tyr-69 mutants—Asymmetric Y69F mutants were generated by PCR site-directed mutagenesis as previously described (10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar). Correct mutations were confirmed by DNA sequencing and the DNA maintained in E. coli STBLII cells to avoid recombination problems (15Strader M.B. Howell E.E. Gibco-BRL Focus. 1997; 19: 24-25Google Scholar). Y69T, Y69K, Y69Q, and Y69L homotetrameric mutants were also generated by PCR-based site-directed mutagenesis. None of the cell lines transformed with these mutants were able to grow in the presence of 20 μg trimethoprim/ml. Because high protein expression was noted for the Y69L mutant, it was pursued. Protein Purification and Characterization—Proteins were purified as previously described (10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar). The asymmetric Y69F mutants were not as prone to aggregation as the asymmetric K32M mutants (companion paper, Hicks et al. (42Hicks S.N. Smiley R.D. Stinnett L.G. Minor K.H. Howell E.E. J. Biol. Chem. 2004; 279: 46995-47002Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar)); however, visible turbidity was sometimes noted at the high protein concentrations (∼100 μm) used in isothermal titration calorimetry experiments. Thus 0.1 g/liter polyethylene glycol 3350 was added to minimize aggregation as well as increase purification yields of the asymmetric mutants (16de Bernandez Clark E. Schwartz E. Rudolph R. Methods Enzymol. 1999; 309: 217-235Crossref PubMed Scopus (223) Google Scholar). Steady-state kinetics were performed for each of the mutants to determine the corresponding Km and kcat values as previously described (Ref. 12Strader M.B. Chopra S. Jackson M. Smiley R.D. Stinnett L.G. Wu J. Howell E.E. Biochemistry. 2004; 43: 7403-7412Crossref PubMed Scopus (19) Google Scholar and Hicks et al. (42Hicks S.N. Smiley R.D. Stinnett L.G. Minor K.H. Howell E.E. J. Biol. Chem. 2004; 279: 46995-47002Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar)). Isothermal Titration Calorimetry—Isotherms were generated for substrate or cofactor binding to each mutant on a MicroCal VP isothermal titration calorimeter at 28 °C as described previously (5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholar, 10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar). MTH polybuffer (50 mm MES plus 100 mm Tris, plus 50 mm acetic acid; pH 8 (17Ellis K.J. Morrison J.F. Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (652) Google Scholar)) was used for NADPH binding studies, whereas 10 mm Tris plus 1 mm EDTA buffer, pH 8, was used for DHF binding studies. Because DHF is a weak acid, the pH of the DHF solution was titrated to pH 8 prior to ligand binding studies. The data for NADPH binding to the mutants were analyzed by Origin software (Version 5.0) using both the single sites model and the sequential sites model where the stoichiometry was set to two. pH Titrations—Homotetrameric R67 DHFR dissociates into dimers upon titration with acid due to protonation of symmetry-related histidine 62 residues (18Nichols R. Weaver C.D. Eisenstein E. Blakley R.L. Appleman J. Huang T.H. Huang F.Y. Howell E.E. Biochemistry. 1993; 32: 1695-1706Crossref PubMed Scopus (35) Google Scholar, 19Park H. Zhuang P. Nichols R. Howell E.E. J. Biol. Chem. 1997; 272: 2252-2258Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This equilibrium is described by,T+2nH+⇆Koverall2DHnREACTION 1 where T is tetramer, DHn is protonated dimer, n is the number of protons, and Koverall equals [T] [H]2n/[DHn]2 and can be described by Ka2n/Kd. This dissociation process can be monitored by a change in fluorescence of symmetry-related tryptophan residues, which become solvent-exposed upon dissociation of the tetramer (18Nichols R. Weaver C.D. Eisenstein E. Blakley R.L. Appleman J. Huang T.H. Huang F.Y. Howell E.E. Biochemistry. 1993; 32: 1695-1706Crossref PubMed Scopus (35) Google Scholar, 19Park H. Zhuang P. Nichols R. Howell E.E. J. Biol. Chem. 1997; 272: 2252-2258Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 20West F.W. Seo H.S. Bradrick T.D. Howell E.E. Biochemistry. 2000; 39: 3678-3689Crossref PubMed Scopus (16) Google Scholar). To determine if Y69L mutations in the homotetramer affect this equilibrium, pH versus fluorescence intensity profiles were generated. Data were fit as described previously by non-linear regression using SAS (18Nichols R. Weaver C.D. Eisenstein E. Blakley R.L. Appleman J. Huang T.H. Huang F.Y. Howell E.E. Biochemistry. 1993; 32: 1695-1706Crossref PubMed Scopus (35) Google Scholar). Although the Y69F asymmetric mutants are unable to undergo a dimer to tetramer equilibrium, because the domains in Quad3 are covalently linked, it is possible for the domains to “open up” or “splay apart” upon titration of symmetry-related histidine 62 residues (10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar, 11Bradrick T.D. Shattuck C. Strader M.B. Wicker C. Eisenstein E. Howell E.E. J. Biol. Chem. 1996; 271: 28031-28037Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This process can also be monitored as a change in tryptophan fluorescence with decreasing pH. To determine whether Y69F asymmetric mutations had affected this process, pH versus fluorescence intensity profiles were generated. Data were fit to a simple ionization equation using SigmaPlot (21Fersht A. Structure and Mechanism in Protein Science. 2nd Ed. W. H. Freeman and Company, New York1999: 101-105Google Scholar). Nomenclature—The naming system used to name the asymmetric mutants follows the pattern established by our companion paper (Hicks et al. (42Hicks S.N. Smiley R.D. Stinnett L.G. Minor K.H. Howell E.E. J. Biol. Chem. 2004; 279: 46995-47002Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar)). Briefly, the Y69F mutation is listed followed by the number indicating the gene copy, which carries the mutation. For example, a single Y69F:1 mutant was constructed where the mutation was placed in gene copy 1. Three double mutants, one triple mutant and one quadruple mutant were also constructed. The double mutants are non-equivalent; this can be seen using the crystal structure for homotetrameric R67 DHFR (Fig. 1, B–D). A model of the NADPH·folate ternary complex that is consistent with NMR and crystallography constraints is shown in Fig. 1E along with the relative positions of Tyr-69, Lys-32, Gln-67, and Ile-68 residues (2Narayana N. Matthews D.A. Howell E.E. Nguyen-huu X. Nat. Struct. Biol. 1995; 2: 1018-1025Crossref PubMed Scopus (76) Google Scholar, 6Li D. Levy L.A. Gabel S.A. Lebetkin M.S. DeRose E.F. Wall M.J. Howell E.E. London R.E. Biochemistry. 2001; 40: 4242-4252Crossref PubMed Scopus (44) Google Scholar, 13Howell E.E. Shukla U. Hicks S.N. Smiley R.D. Kuhn L.A. Zavodszky M.I. J. Comput. Aided Mol. Des. 2001; 15: 1035-1052Crossref PubMed Scopus (20) Google Scholar, 14Pitcher 3rd, W.H. DeRose E.F. Mueller G.A. Howell E.E. London R.E. Biochemistry. 2003; 42: 11150-11160Crossref PubMed Scopus (19) Google Scholar). In addition to Y69F asymmetric mutants, the effects of Y69L mutations in the homotetrameric enzyme were also assessed. This construct is named Y69L R67 DHFR and contains four symmetry-related Y69L mutations. Steady-state Kinetics—To gain a better understanding of the role of Tyr-69 in binding and catalysis, steady-state kinetic data were collected for each of the asymmetric Y69F mutants and are presented in Table I. In general, the Km values for NADPH and DHF as well as the kcat display a trend contingent upon the location and number of Y69F mutations. A wild-type Km (NADPH) value is observed for both the Y69F:1 and Y69F: 1+3 mutants, whereas the Km (NADPH) for the Y69F:1+2 and Y69F:1+4 mutants is ∼3- to 4-fold weaker. The Km (NADPH) values for the triple mutant and quadruple mutants continue to increase. The kinetic parameters for the Y69F:1+2+3+4 mutant are similar to those for Y69F R67 DHFR (9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar). Thus, for NADPH binding interactions in the productive, ternary complex, as the number of mutations increases, Km (NADPH) concurrently increases. The two exceptions to this trend are the Y69F:1 and Y69F:1+3 mutants, which both have wild-type Km values.Table IComparison of kinetic parameters for wild-type R67 DHFR, Quad3 DHFR, Y69F asymmetric mutants, Y69L R67 DHFR, and Y69F R67 DHFR at pH 7.0Enzyme variantkcatKm (NADPH)Km (DHF)s-1μmWT R67 DHFRaTaken from Ref. 391.3 ± 0.13.0 ± 0.15.8 ± 0.1Quad3 DHFRbTaken from Ref. 100.81 ± 0.024.4 ± 0.46.7 ± 0.4Y69F:1 DHFR0.98 ± 0.066.4 ± 0.621 ± 2Y69F:1+2 DHFR0.96 ± 0.0114 ± 0.820 ± 0.9Y69F:1+3 DHFR0.56 ± 0.013.1 ± 0.420 ± 2Y69F:1+4 DHFR1.9 ± 0.0814 ± 128 ± 2Y69F:1+2+3 DHFR1.4 ± 0.0421 ± 135 ± 2Y69F:1+2+3+4 DHFR1.4 ± 0.0440 ± 354 ± 3Y69F R67 DHFRcRefit from Ref. 9 using the non-linear, global SAS fit described in Ref. 102.9 ± 0.169 ± 368 ± 4Y69L R67 DHFR0.16 ± 0.0168 ± 3180 ± 11Y69H R67 DHFRcRefit from Ref. 9 using the non-linear, global SAS fit described in Ref. 100.014 ± 0.002176 ± 6.046 ± 4.5a Taken from Ref. 39Reece L.J. Nichols R. Ogden R.C. Howell E.E. Biochemistry. 1991; 30: 10895-10904Crossref PubMed Scopus (55) Google Scholarb Taken from Ref. 10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholarc Refit from Ref. 9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar using the non-linear, global SAS fit described in Ref. 10Smiley R.D. Stinnett L.G. Saxton A.M. Howell E.E. Biochemistry. 2002; 41: 15664-15675Crossref PubMed Scopus (15) Google Scholar Open table in a new tab Similar to NADPH interactions in the ternary complex, Km (DHF) values also tend to increase as the number of mutations is increased. Specifically, the Km (DHF) for the single and all double mutants is ∼2- to 3-fold weaker than the Km (DHF) for Quad3. In addition, the Km (DHF) for the Y69F:1+2+3 mutant continues the trend of increasing as additional mutations are added, up to the limit associated with the Y69F:1+2+3+4 mutant. Although binding of both NADPH and DHF in the Michaelis complex is, in general, weakened as the number of Y69F mutations is increased, the kcat value increases over a range of ∼2-fold. A trend is noted where the kcat increases when two Y69F mutations occur at the position that corresponds to one dimer-dimer interface in wt R67 DHFR, as occurs in the Y69F: 1+4 (see Fig. 1D), Y69F:1+2+3, and Y69F:1+2+3+4 mutants, suggesting this topology may be preferred in the transition state. Isothermal Titration Calorimetry—Although steady-state kinetics provide important insight into interactions between ligands in the Michaelis complex, these Km values do not necessarily correspond to dissociation constants, because other events can contribute to the observed Michaelis constants (21Fersht A. Structure and Mechanism in Protein Science. 2nd Ed. W. H. Freeman and Company, New York1999: 101-105Google Scholar). Isothermal titration calorimetry (ITC) provides a direct measure of the heat exchange upon ligand binding as well as a direct measure of the Kd for the ligand of interest (22Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar, 23Leavitt S. Freire E. Curr. Opin. Struct. Biol. 2001; 11: 560-566Crossref PubMed Scopus (560) Google Scholar). Thus ITC experiments were performed using binary complex conditions where either two NADPH molecules or two DHF molecules can interact with the enzyme. Data for NADPH binding to the Y69F asymmetric mutants are summarized in Table II. These data were analyzed using both the single sites model and the sequential sites model where the stoichiometry was set to two. The Kd and ΔH values for the first NADPH binding event were similar regardless of the model used to describe the data. However the Kd2 and ΔH2 values generated using the sequential sites model varied, most likely because the Kd2 values are high and fall outside of the detection window afforded by the calorimeter. For accurate results, the c value, where c equals [protein]*Ka, should fall within 1 and 1000 (22Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar, 23Leavitt S. Freire E. Curr. Opin. Struct. Biol. 2001; 11: 560-566Crossref PubMed Scopus (560) Google Scholar). Therefore, only the values for the first NADPH binding event are reported. For some mutants (i.e. Y69F:1+2), low stoichiometries were observed and may reflect the presence of some inactive protein in the reaction solution and/or concentration measurement errors.Table IIComparison of NADPH binding constants for Y69F asymmetric mutants and Y69L R67 DHFR as determined by isothermal titration calorimetry Data reported were fit using the single sites model. Each value is an average of at least two different experiments.ComplexKdΔHStoichiometryμmcal/moleQuad3 DHFR8 ± 0.3-7600 ± 2600.83 ± 0.01Y69F:1 DHFR10 ± 1-6200 ± 1200.88 ± 0.003Y69F:1+2 DHFR25 ± 2-4900 ± 1700.69 ± 0.02Y69F:1+3 DHFR15 ± 1-5200 ± 3900.72 ± 0.01Y69F:1+4 DHFR9 ± 1-7500 ± 4900.84 ± 0.02Y69F:1+2+3 DHFR25 ± 2-5000 ± 1200.75 ± 0.03Y69F:1+2+3+4 DHFR52 ± 5-2400 ± 2100.95 ± 0.11Wt R67 DHFRaValue describing the first binding site in a two sites model, from Ref. 55.0 ± 0.3-8600 ± 2001.56 ± 0.14Y69F R67 DHFRbRefit from Ref. 9 to a single sites model65 ± 6-2200 ± 1701.1 ± 0.1Y69L R67 DHFR75 ± 0.4-2800 ± 401.1 ± 0.1a Value describing the first binding site in a two sites model, from Ref. 5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholarb Refit from Ref. 9Strader M.B. Smiley R.D. Stinnett L.G. VerBerkmoes N.C. Howell E.E. Biochemistry. 2001; 40: 11344-11352Crossref PubMed Scopus (28) Google Scholar to a single sites model Open table in a new tab In general, the effects of the Y69F mutation(s) on NADPH binding to the single and double mutants were minor, with the Y69F:1+2 configuration (see Fig. 1B) having the largest effect (∼3-fold). As three to four mutations were added, the Kd values correspondingly increased (up to a 6.5-fold effect). The enthalpy change for NADPH binding to the Y69F:1 and Y69F:1+4 mutants was similar to that for Quad3, whereas the ΔH became less negative as the number of mutations was increased in the Y69F:1+2+3 and Y69F:1+2+3+4 mutants. Isothermal titration calorimetry experiments were also performed to determine the effects of asymmetric Y69F mutations on interactions with DHF. Previously, isotherms generated for DHF binding to wild-type R67 DHFR were fit to an interacting sites model, because isotherms displayed a “hook” reflecting positive cooperativity between two bound DHF molecules (5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholar). (A stoichiometry of two was previously confirmed by time-resolved fluorescence anisotropy measurements (5Bradrick T.D. Beechem J.M. Howell E.E. Biochemistry. 1996; 35: 11414-11424Crossref PubMed Scopus (54) Google Scholar).) New calorimeters have increased sensitivity to minor changes in solution pH during the titration. Close attention to the pH of both the ligand and protein solution indicated that the prominence of the hook is related to changes in pH of the protein solution upon injection of ligand into the solution. Additional studies indicated that the presence of the hook is affected by the ionic strength of the buffering solution (data not shown). The sensitivity of DHF binding to R67 DHFR may also be related to previous NMR studies that found DHF dimerizes in solution and this dimerization is affected by ligand concentration, pH, and ionic strength (24Poe M. J. Biol. Chem. 1973; 248: 7025-7032Abstract Full Text PDF PubMed Google Scholar, 25Khaled M.A. Krumdieck C.L. Biochem. Biophys. Res. Commun. 1985; 130: 1273-1280Crossref PubMed Scopus (18) Google Scholar). Due to the characteristics of the isotherms generated for DHF binding to the Y69F asymmetric mutants, we are unable to consistently fit the data using the Origin software provided by MicroCal. In particular, if there are only a few points in the “hook,” the Origin software does not provide a strong weighting to those points and the fit line excludes them. Therefore, for comparative purposes, we have overlaid the raw data for DHF binding to each of the asymmetric mutants in Fig. 2A. Based on the raw data, a trend is observed in both the shape of the curve and the initial heat released upon ligand binding as well as the prominence of the hook. Specifically, there is a trend in the DHF concentration required to reach saturation for the asymmetric mutants. The signal for Quad3 increases most dramatically as the DHF concentration is increased, whereas the steepness of the slope in the titrations decreases as the number of mutations is increased. This suggests that, as the number of mutations is
Referência(s)