Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.     

Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

The α-Helix of Ribonuclease T1as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability

Measurements of the change in conformational stability, Δ(ΔG), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T₁have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T₁behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin. In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T. (1998) J. Biol. Chem. 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(364), and Lys(372)) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo. All mutant proteins maintained activities for DNA replication and ATP binding. A mutant protein in which Lys(372) was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins. Thus, Lys(372) seems to play an important role in the interaction between DnaA protein and acidic phospholipids. Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)--putidaredoxin: roles of glutamine 360 at the heme proximal site

We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Leu. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K(m)) that were moderately perturbed for the Lys and Leu mutants and the distinctly increased for the Glu mutant. Although the alterations in K(m) indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Glu mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Glu mutant but not in the Lys and Leu mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe(3+)/Fe(2+) couple by 20-70 mV. Since the amide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450(cam) by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg112.     

Competitive inhibition of electron donation to photosystem 1 by metal-substituted plastocyanin

The electron transfer from wild-type spinach plastocyanin (Pc) to photosystem 1 has been studied by flash-induced absorption changes at 830 nm. The decay kinetics of photo-oxidized P700 are drastically slower in the presence of Ag(I)-substituted Pc, while addition of Zn(II)-substituted Pc has a weaker effect. The metal-substituted forms of Pc act as competitive inhibitors of the reaction between normal, Cu-containing, Pc and P700. The inhibition constants obtained from an analysis of the kinetic data were 30 and 410 muM for Ag(I)- and Zn(II)-substituted Pc, respectively. When the Gly8Asp mutant form of Pc was used instead of the wild-type form, the corresponding values were found to be 77 and 442 muM. If the Ag- and Zn-derivatives can be considered as structural mimics of reduced and oxidized CuPc, respectively, our results imply that there is a redox-induced decrease in the affinity between Pc and photosystem 1 that follows the electron donation to P700. Our data also imply that the Gly8Asp mutation can diminish the magnitude of this change. The findings reported here are consistent with a reaction mechanism where the electron transfer in the complex between Pc and photosystem 1 is assumed to be reversible.     

Interaction between mitochondrial CYP27B1 and adrenodoxin: role of arginine 458 of mouse CYP27B1

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.     

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.     

Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine

Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.     

Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor

The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change.     

Kinetic and structural analysis of active site mutants of monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae

5,10-Methylenetetrahydrofolate dehydrogenase (MTD) catalyzes the reversible oxidation of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate. This reaction is critical for the supply of one-carbon units at the required oxidation states for the synthesis of purines and dTMP. For most MTDs, dehydrogenase activity is co-located with a methenyl-THF cyclohydrolase activity as part of bifunctional or trifunctional enzyme. The yeast Saccharomyces cerevisiae contains a monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase (yMTD). Kinetic, crystallographic, and mutagenesis studies were conducted to identify critical residues in order to gain further insight into the reaction mechanism of this enzyme and its apparent lack of cyclohydrolase activity. Hydride transfer was found to be rate-limiting for the oxidation of methylenetetrahydrofolate by kinetic isotope experiments (V(H)/V(D) = 3.3), and the facial selectivity of the hydride transfer to NAD(+) was determined to be Pro-R (A-specific). Model building based on the previously solved structure of yMTD with bound NAD cofactor suggested a possible role for three conserved amino acids in substrate binding or catalysis: Glu121, Cys150, and Thr151. Steady-state kinetic measurements of mutant enzymes demonstrated that Glu121 and Cys150 were essential for dehydrogenase activity, whereas Thr151 allowed some substitution. Our results are consistent with a key role for Glu121 in correctly binding the folate substrate; however, the exact role of C150 is unclear. Single mutants Thr57Lys and Tyr98Gln and double mutant T57K/Y98Q were prepared to test the hypothesis that the lack of cyclohydrolase activity in yMTD was due to the substitution of a conserved Lys/Gln pair found in bifunctional MTDs. Each mutant retained dehydrogenase activity, but no cyclohydrolase activity was detected.     

Redox-dependent structural changes in the superoxide reductase from Desulfoarculus baarsii and Treponema pallidum: a FTIR study

The redox-induced structural changes at the active site of the superoxide reductase (SOR) from Desulfoarculus baarsii and Treponema pallidum have been monitored by means of FTIR difference spectroscopy coupled to electrochemistry. With this technique, the structure and interactions formed by individual amino acids at a redox site can be detected. The infrared data on wild-type, Glu47Ala, and Lys48Ile mutants of the SOR from D. baarsii provide experimental support for the conclusion that the two different coordination motifs observed in the three-dimensional structure of the SOR from Pyrococcus furiosus [Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. (2000) Biochemistry 39, 2499-2508] correspond to the two redox forms of the SOR iron center. We extend this result to the center II iron of SOR of the desulfoferrodoxin type. Similar structural changes are also observed upon iron oxidation in the SOR of T. pallidum. In D. baarsii, the IR modes of the Glu47 side chain support that it provides a monodentate ligand to the oxidized iron, while it does not interact with Fe(2+). Structural changes at the level of peptide bond(s) observed upon iron oxidation in wild-type are suppressed in the Glu47Ala mutant. We propose that the presence of the Glu side chain plays an important role for the structural reorganization accompanying iron oxidation. We identified the infrared modes of the Lys48 side chain and found that a change in its environment occurs upon iron oxidation. The lack of other structural changes upon the Lys48Ile mutation shows that the catalytic role of Lys, as evidenced by pulse radiolysis experiments [Lombard, M., Houée-Levin, C., Touati, D., Fontecave, M., and Nivière, V. (2001) Biochemistry 40, 5032-5040], is purely electrostatic, guiding superoxide toward the reduced iron.