Electron spin echo modulation and nuclear relaxation studies of staphylococcal nuclease and its metal-coordinating mutants

Electron spin echo envelope modulation (ESEEM) spectroscopy has been applied to the determination of the number of water molecules coordinated to the metal in the binary complex of staphylococcal nuclease with Mn2+, to the ternary enzyme-Mn2+-3',5'-pdTp complex, and to ternary complexes of a number of mutant enzymes in which metal-binding ligands have been individually altered. Quantitation of coordinated water is based on ESEEM spectral comparisons of Mn2+-EDTA and Mn2+-DTPA, which differ by a single inner sphere water, and with Mn2+-(H2O)6. It was found that Mn2+ in the ternary complex of the wild-type enzyme has a single additional coordinated water, as compared to Mn2+ in the binary complex, confirming earlier findings based on T1 measurements of bound water [Serpersu, E. H., Shortle, D. L., & Mildvan, A. S. (1987) Biochemistry 26, 1289-1300]. Ternary complexes of the mutant proteins D40E, D40G, and D21Y have the same number of water ligands as the ternary complex of the wild-type enzyme, while the D21E mutant has one less water ligand. In order to maintain octahedral coordination geometry, these findings require two additional ligands to Mn2+ from the protein in the binary complex of the wild-type enzyme, probably Glu 43 and Asp 19, and one additional ligand from the protein in the ternary D40G and D21E complexes. Other ESEEM studies of ternary Mn2+ complexes of wild-type, D21E, and D21Y mutants indicate the coordination by Mn2+ of a phosphate of 3',5'-pdTp, as demonstrated by a 31P contact interaction of 3.9 +/- 0.3 MHz. Magnetic interaction of Mn2+ with 31P could not be demonstrated with the D40G and D40E mutants, suggesting that metal-phosphate distances are greater in these mutants than in the wild-type protein. In a parallel NMR study of the paramagnetic effects of enzyme-bound Co2+ (which occupies the Mn2+ site on the enzyme) on the T1 of 31P from enzyme-bound 3',5'-pdTp and 5'-TMP, it was found that metal to 5'-phosphate distances are 0.9-1.6 A shorter in ternary complexes of the wild-type enzyme and of the D21E mutant than in ternary complexes of the D40G mutant. In all cases, the 5'-phosphate of pdTp is in the inner coordination sphere of Co2+ and the 3'-phosphate is predominantly in the second coordination sphere.     

Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: factors contributing to catalysis

To determine the origin of the overall approximately 10(16)-fold rate enhancement of DNA hydrolysis catalyzed by staphylococcal nuclease, the effects of single mutations that alter the amino acid residue at each of the essential positions Asp-21, Asp-40, Thr-41, Arg-35, and Arg-87 have been examined. Metal ion and substrate analogue binding were quantitated by EPR, by the paramagnetic effects of Mn2+ on 1/T1 of water protons, and by fluorescence titrations, yielding the six dissociation constants of the ternary enzyme-Mn2+-3',5'-pdTp and enzyme-Ca2+-3',5'-pdTp complexes. The kinetic parameters kcat, KACa, KMCa, KSDNA, KMDNA, and KIMn were determined by monitoring the rate of DNA hydrolysis. By thermodynamic and kinetic criteria, Mn2+ binds tightly to the Ca2+ binding site of the enzyme but is at least 36,000-fold less effective than Ca2+ in activating the enzyme. Alterations of the liganding residues in the D40G, D40E, T41P, D21E, and D21Y mutants generally weaken the binding of Ca2+ less than or equal to 12.7-fold and of Mn2+ less than or equal to 5.4-fold, exert little effect on the KSDNA or KMDNA (less than or equal to 3.2-fold), and raise the affinity of the enzyme and its metal complexes for 3',5'-pdTp by factors less than or equal to 13.5-fold. Small changes in the ligand geometry are also reflected in the Mn2+ complexes of the liganding mutants (i.e., those in which the metal-liganding amino acids have been altered) by decreases in the electron-spin relaxation time of Mn2+. Inhibitory effects on kcat are noted in all of the liganding mutants with D40E, D40G, T41P, D21E, and D21Y showing 12-, 30-, 37-, 1500-, and greater than or equal to 29,000-fold reductions, respectively. The greater than or equal 10(3)-fold larger inhibitory effects on kcat of enlarging Asp-21 as compared to enlarging Asp-40 are ascribed to the displacement of the adjacent water molecule which attacks the phosphodiester. Mutations of each of the essential Arg residues to Gly (R35G and R87G) reduce kcat by factors greater than or equal to 35,000 but weaken metal binding less than or equal to 9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic and magnetic resonance studies of the glutamate-43 to serine mutant of staphylococcal nuclease

The Glu-43 residue of staphylococcal nuclease has been proposed to function as a general base that facilitates the attack of water on the phosphodiester substrate [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. With DNA as substrate, Vmax in the glutamate-43--serine (E43S) mutant enzyme is decreased by 2700-fold at pH 7.4 but only 376-fold at pH 9.9. With the wild-type enzyme, Vmax increases with pH to pH 9.2, above which it becomes less sensitive to further increase in pH, leveling off at pH 9.8. In contrast, Vmax of the E43S mutant continues to rise, first order in [OH-], to pH 9.8. Above pH 10 both activities fall irreversible. Hence the hydroxyl ion can partially replace the effect of Glu-43 on kcat, in accord with the proposed role of Glu-43 as a general base. The inflection point in the curve relating pH to log Vmax of the wild-type enzyme at pH 9.4 may reflect the ionization of a Ca2+-bound water, or of a Lys or Tyr residue at the active site. The activator Ca2+ and the competitive inhibitor Mn2+ bind to the E43S mutant an order of magnitude more weakly than to the wild-type enzyme as detected by kinetics and by direct metal binding studies, and approximately one additional water ligand on Mn2+ is found in the binary Mn2+ complex of the E43S mutant (1.4 +/- 0.2) as compared to that of the wild-type enzyme (0.8 +/- 0.2). These data suggest that Glu-43 coordinates the divalent cation in the binary enzyme-metal complex but dissociates from the metal to create a water binding site and to function as a general base in the ternary enzyme-metal-DNA complex. While a 2-fold weaker binding of DNA to the Ca2+ complex of the E43S mutant than to the wild-type enzyme is found by kinetic studies, an order of magnitude tighter binding of the competitive inhibitor 3',5'-pdTp to the Mn2+ and Ca2+ complexes of E43S is found by direct binding studies. Distances from Co2+ to phosphorus in the ternary enzyme-Co2+-pdTp complexes reveal coordination of only the 5'-phosphate by Co2+ on the wild-type enzyme but coordination of both the 3'- and 5'-phosphates of pdTp on the E43S mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.

In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.     

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.     

Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.     

Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.

Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical DeltaDeltaG degrees bind values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+. In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. DeltaDeltaG degrees bind values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

NMR docking of a substrate into the X-ray structure of staphylococcal nuclease.

The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.     

The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phospholipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.     

Role of the proposed pore-forming segment of the Ca2+ release channel (ryanodine receptor) in ryanodine interaction

In earlier studies we showed that point mutations introduced into the proposed pore-forming segment, GVRAGGGIGD (amino acids 4820-4829), of the mouse cardiac ryanodine receptor reduced or abolished high affinity [3H]ryanodine binding. Here we investigate the effects of these mutations on the affinity and dissociation properties of [3H]ryanodine binding and on ryanodine modification of the ryanodine receptor channel at the single channel and whole cell levels. Scatchard analysis and dissociation studies reveal that mutation G4824A decreases the equilibrium dissociation constant (K(d)) and the dissociation rate constant (k(off)), whereas mutations G4828A and D4829A increase the K(d) and k(off) values. The effect of ryanodine on single G4828A and D4829A mutant channels is reversible on the time scale of single channel experiments, in contrast to the irreversible effect of ryanodine on single wild-type channels. Ryanodine alone is able to induce a large and sustained Ca2+ release in HEK293 cells transfected with the R4822A or G4825A mutant cDNA at the resting cytoplasmic Ca2+ but causes little or no Ca2+ release in cells transfected with the wild-type cDNA. Mutation G4826C diminishes the functional effect of ryanodine on Ca2+ release but spares caffeine-induced Ca2+ release in HEK293 cells. Co-expression of the wild-type and G4826C mutant proteins produces single channels that interact with ryanodine reversibly and display altered conductance and ryanodine response. These results are consistent with the view that the proposed pore-forming segment is a critical determinant of ryanodine interaction. A putative model of ryanodine-ryanodine receptor interaction is proposed.     

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.     

Investigation of conserved acidic residues in 3-hydroxy-3-methylglutaryl-CoA lyase: implications for human disease and for functional roles in a family of related proteins

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metal-dependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280. HMG-CoA lyase deficiency results from a human mutation that substitutes lysine for glutamate 279. The E279K mutation has also been engineered; expression in Escherichia coli produces an unstable protein. Substitution of alanine for glutamate 279 produces a protein that is sufficiently stable for isolation and retains substantial catalytic activity. However, thermal inactivation experiments demonstrate that E279A is much less stable than wild-type enzyme. HMG-CoA lyase deficiency also results from mutations at aspartate 42. Substitutions that eliminate a carboxyl group at residue 42 perturb cation binding and substantially lower catalytic efficiency (104-105-fold decreases in specific activity for D42A, D42G, or D42H versus wild-type). Substitutions of alanine for the other conserved acidic residues indicate the importance of glutamate 72. E72A exhibits a 200-fold decrease in kcat and >103-fold decrease in kcat/Km. E72A is also characterized by inflation in the Km for activator cation (26-fold for Mg2+; >200-fold for Mn2+). Similar, but less pronounced, effects are measured for the D204A mutant. E72A and D204A mutant proteins both bind stoichiometric amounts of Mn2+, but D204A exhibits only a 2-fold inflation in KD for Mn2+, whereas E72A exhibits a 12-fold inflation in KD (23 microm) in comparison with wild-type enzyme (KD = 1.9 microm). Acidic residues corresponding to HMG-CoA lyase Asp-42 and Glu-72 are conserved in the HMG-CoA lyase protein family, which includes proteins that utilize acetyl-CoA in aldol condensations. These related reactions may require an activator cation that binds to the corresponding acidic residues in this protein family.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme

In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

The kinetics of Ca(2+)-dependent switching in a calmodulin-IQ domain complex

We have performed a kinetic analysis of Ca2+-dependent switching in the complex between calmodulin (CaM) and the IQ domain from neuromodulin, and have developed detailed kinetic models for this process. Our results indicate that the affinity of the C-ter Ca2+-binding sites in bound CaM is reduced due to a approximately 10-fold decrease in the Ca2+ association rate, while the affinity of the N-ter Ca2+-binding sites is increased due to a approximately 3-fold decrease in the Ca2+ dissociation rate. Although the Ca2+-free and Ca2+-saturated forms of the CaM-IQ domain complex have identical affinities, CaM dissociates approximately 100 times faster in the presence of Ca2+. Furthermore, under these conditions CaM can be transferred to the CaM-binding domain from CaM kinase II via a ternary complex. These properties are consistent with the hypothesis that CaM bound to neuromodulin comprises a localized store that can be efficiently delivered to neuronal proteins in its Ca2+-bound form in response to a Ca2+ signal.     

Effect of Hailey-Hailey Disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1)

ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1 approximately P --> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.