Aspartate Side Chain Research Articles

Ion channels of biological membranes: prediction of single channel conductance

The Poisson-Boltzmann equation was solved numerically for models of the pore regions of the Shaker K+ channel and of two glycoporins (LamB and ScrY) to yield electrostatic potential profiles along the pore axes. From these potential profiles, single-channel current-voltage (I–V) relations were calculated. The importance of a proper treatment of the ionisation state of two rings of aspartate sidechains at the mouth of the K+ channel pore emerged from such calculations. The calculated most likely state, in which only two of the eight aspartate sidechains were deprotonated, yielded better agreement with experimental conductance data. An approximate calculation of single-channel conductances based simply on pore geometry yielded very similar conductance values for the two glycoporins. This differed from an␣experimentally determined conductance ratio of ScrY:LamB=10:1. Preliminary electrostatics calculations appeared to reproduce the observed difference in conductance between the two glycoporins, confirming that single-channel conductance is determined by electrostatic as well as geometric considerations.

Crystal Structures of Catalytic Core Domains of Retroviral Integrases and Role of Divalent Cations in Enzymatic Activity

Crystal structures of the enzymatically competent catalytic domains of HIV-1 and ASV IN have been solved in the last few years. The structure of HIV-1 IN has been described only for apoenzyme and for a complex with Mg2+, whereas the structure of ASV IN has been presented as the apoenzyme, in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Zn2+, and Cd2+), and with an inhibitor. A single ion of Mn2+, Mg2+, or Ca2+ interacts with the two aspartate side chains of the D,D(35)E catalytic center in octahedral coordination with four water molecules. However, two ions of Zn2+ or Cd2+ bind to the active site of IN with tetrahedral and octahedral coordination, respectively. Only small adjustments take place in the active site of ASV IN on binding of the metal cofactor(s), which are absolutely required for the activity of this enzyme. The placement of the side chains and metal ions in the active site is very similar to that observed even in distant members of this superfamily of polynucleotidyltransferases. Here the role of divalent cations in the enzymatic activity of IN and the search for inhibitors of this enzyme are discussed.

Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change?

C2 domains are widespread Ca2+-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C2A domain of synaptotagmin I is believed to play a key role in neurotransmitter release through its Ca2+-dependent interactions with syntaxin and phospholipids. Elucidating the structural consequences of Ca2+ binding to the C2A domain is critical for understanding its mechanism of action and for models of the functions of other C2 domains. We have determined the solution structure of the Ca2+-free and Ca2+-bound forms of the C2A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C2 domain in its Ca2+-free and Ca2+-bound forms. Three Ca2+ ions were included in the Ca2+-bound structure, yielding a Ca2+-binding motif that involves five aspartate side chains and one serine side chain. Ca2+ immobilizes the structure of the C2A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C2A domain of synaptotagmin I is different from that used by Ca2+-binding proteins of the EF-hand family. The main effect of Ca2+ binding on the C2A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C2A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C2A domain and the PLC-delta1 C2 domain suggests that the latter binds four Ca2+ ions and acts by a similar mechanism. This mechanism may also be valid for other C2 domains that share the unusual ability to bind multiple Ca2+ ions in a tight cluster at the tip of the domain.

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.

Energetics of polar side-chain interactions in helical peptides: salt effects on ion pairs and hydrogen bonds.

The energetics of the interaction between the polar side chains of glutamate or aspartate with lysine and glutamate with histidine have been determined using a model alanine-based peptide helix. An evaluation of the effects of NaCl and pH on the interactions between these acidic and basic residues in several different orientations and spacings in an alpha-helical peptide has been made. For many of the peptides, we find a considerable interaction between the polar side chains. In general, the shorter side chains show stronger interactions, but there are more restrictions on the precise geometry of the interactions as dictated by the spacing and orientation of the polar residues in the alpha-helical peptide. The energetics of the interaction between the fully-charged ion pairs can be diminished by added salt, but the interaction is not completely screened even at 2.5 M NaCl. The strength of the interaction between a charged and neutral side chain is not as sensitive to the ionic strength of the solution, suggesting that solvent-exposed hydrogen bonds are forming. All the interactions between the polar residues employed here stabilize helix formation, suggesting that solvent-exposed ion pairs and hydrogen bonds can contribute to the conformational stability of proteins and peptides.

Functional identification of calcium binding residues in bovine alpha-lactalbumin.

The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.

Molecular Determinants of Arg-Gly-Asp Ligand Specificity for β3 Integrins

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alphaIIbbeta3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alphaIIbbeta3 versus the vitronectin receptor alphaVbeta3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alphaIIbbeta3 and not at all to any other RGD-cognitive integrin, particularly alphaVbeta3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alphaIIbbeta3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alphaVbeta3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alphaVbeta3 in a cation-dependent manner, but does not bind at all to alphaIIbbeta3 under any conditions. AP7.4 binding to alphaVbeta3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alphaVbeta3. Despite these differences in specificity for alphaIIbbeta3 and alphaVbeta3, AP7 and AP7.4 remain selective for the beta3 integrins and do not bind to cell lines that express the RGD-cognitive integrins alphaVbeta5 or alpha5beta1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on beta3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.

Investigation of substrate activation by 4-chlorobenzoyl-coenzyme A dehalogenase.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA), using the carboxylate side chain of aspartate 145 to displace the chloride from C(4) of the benzoyl ring. Previous UV-visible, Raman, and 13C NMR studies of enzyme-bound substrate analog or product ligand indicated that the environment of the enzyme active site induces a significant reorganization of the benzoyl ring pi-electrons. This observation was interpreted as evidence for electrophilic catalysis [viz. active-site-induced polarization of electron density away from the ring C(4)] [Taylor, K. L., Liu, R.-Q., Liang, P.-H., Price, J., Dunaway-Mariano, D., Tonge, P. J., Clarkson, J., & Carey, P. R. (1995) Biochemistry 34, 13881]. The recent crystal structure of the dehalogenase-4-HBA-CoA complex reveals two hydrogen bonds contributed to the benzoyl C=O by the backbone amide protons of Gly114 and Phe64 and a possible dipolar interaction with the positive pole of the 114-121 alpha-helix. Residues closely surrounding the benzoyl ring include W137, D145, W89, F64, F82, and H90. In the present study, the mutants D145A, H90Q, W137F, W89F, W89Y, F64L, F82L, and G114A were prepared to examine the effect of amino acid substitution on catalysis and on perturbation of the UV-visible spectral properties of the substrate benzoyl ring. Substitution of the two catalytic residues D145 and H90 inhibited catalysis but not ligand binding or the induction of the red shift in the benzoyl ring absorption. These two residues do not appear to contribute to substrate benzoyl ring binding or polarization. The F64L, F82L, W89F, and W137F mutants retained substantial catalytic activity and the ability to induce the red shift. The W89Y mutant, on the other hand, is inhibited in catalysis and ligand binding, suggesting that hydrophobicity more than packing may be critical for the benzoyl ring binding/activation. The G114A mutant was shown to be strongly inhibited in both substrate binding and activation, indicating that H-bonding and/or interaction with the dipole of the 114-121 alpha-helix may be crucial.

X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate.

The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric molecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.

An eye lens protein-water structure: 1.2 A resolution structure of gammaB-crystallin at 150 K.

gammabeta-crystallin is a structural protein of the eye lens with a role in the maintenance of an even distribution of protein and water over distances around the wavelength of light, preserving lens transparency. The structure of the 174-residue bovine protein has already been determined at room temperature to 1.47 A resolution. By flash freezing the protein crystals, data have now been collected to a nominal resolution limit of 1.2 A as radiation damage was essentially eliminated. The protein-water model has been refined against this data using the program RESTRAIN converging to an R factor of 18.5% with all data. Atomic positions are clearly indicated in the electron-density maps. Discrete bimodal disorder has been visualized for a few side chains. Out of a total of 498 water molecules present in the crystal asymmetric unit, 394 have been modelled and refined at unit occupancy. The solvent structure is extremely well ordered with an average B value of 23.4 A(2). Partially occupied sites have been identified where disorder in the protein induces concomitant disorder in the local solvent structure. The solvent structure covers 97% of the solvent-exposed surface of the protein in the crystal. 126 water molecules are distributed in second and higher hydration shells. There are networks of hydrogen-bonded solvent extending up to 64 molecules in a network, comprising trimers and tetramers as well as five- and six-membered water-ring structures. The hydration of the protein surface is dominated by arginine and aspartate side chains. Extensive cages of highly ordered solvent molecules are also observed around exposed non-polar groups.

Determination of the Gene Sequence and the Three-dimensional Structure at 2.4 Å Resolution of Methanol Dehydrogenase from MethylophilusW3A1

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophusW3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 Å resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca 2+and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cispeptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold β-propeller, each “blade” of which is a four-stranded antiparallel twisted β-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four β-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold β-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight β-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the β-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent β-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca 2+is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.

Kinetic and Structural Consequences of Replacing the Aspartate Bridge by Asparagine in the Catalytic Metal Triad of Escherichia coliAlkaline Phosphatase

In each subunit of the homodimeric enzyme Escherichia colialkaline phosphatase, two of the three metal cofactors, Zn 2+and Mg 2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn 2 +or Mg 2 +. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg 2+at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg 2+increased. Under optimal conditions, the k cat/ K mratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 Å resolution at pH 7.5, and then to 2.1 Å resolution at pH 9.2 with 20 mM MgCl 2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound viaone oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.

Crystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 A resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family.

Mouse lung carbonyl reductase (MLCR) is a member of the short-chain dehydrogenase/reductase (SDR) family. Although it uses both NADPH and NADH as coenzymes, the structural basis of its strong preference for NADPH is unknown. The crystal structure of the ternary complex of MLCR (with NADPH and 2-propanol) has been determined at 1.8 A resolution. This is the first three-dimensional structure of a carbonyl reductase, and MLCR is the first member of the SDR family to be solved in complex with NADPH (rather than NADH). Comparison of the MLCR ternary complex with three structures reported previously for enzymes of the SDR family (all crystallized as complexes with NADH) reveals a pair of basic residues (Lys17 and Arg39) making strong electrostatic interactions with the 2'-phosphate group of NADPH. This pair of residues is well conserved among the NADPH-preferring enzymes of the SDR family, but not among the NADH-preferring enzymes. In the latter, an aspartate side chain occupies the position of the two basic side chains. The aspartate residue, which would come into unacceptably close contact with the 2'-phosphate group of the adenosine moiety of NADPH, is replaced by a threonine or alanine in the primary sequences of NADPH-preferring enzymes of the SDR family. The cofactor preferences exhibited by the enzymes of the SDR family are mainly determined by the electrostatic environment surrounding the 2'-hydroxyl (or phosphate) group of the adenosine ribose moiety of NADH (or NADPH). Thus, positively charged and negatively charged environments correlate with preference for NADPH and NADH respectively.

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations.

Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

A molecular mechanics/continuum reaction field investigation of the interactions between polar amino acid side chains in water and organic solvents

AbstractAn integrated procedure that computes in a consistent way both the intermolecular interaction energies and the solvation energies is reported. It interfaces the Sum of Interactions Between Fragments Ab initio computed molecular mechanics and the Langlei–Claverie continuum reaction field procedures. These two methodologies formulate the interaction energy as a sum of separate electrostatic, polarization, dispersion, and repulsion terms; the first two are computed with the help of distributed charges, dipoles, and quadrupoles derived from the ab initio SCF or MP2 molecular wave fundctions of individual solutes. This computational procedure will be used to estimate the solvent contribution to the interaction energy between polar amino acids side chains. We will consider, on the one hand, the terminal fragments of the side chains of aspartate and glutamate—namely the acetate anion and its protonated counterpart, acetic acid—and on the other hand, the terminal fragments of the cationic residues lysine, histidinium, and arginine, as represented by methylammonium, imidazolium, and methylguanidinium cations, respectively. The deprotonated counterpart of imidazolium, imidazole, is also investigated. With water as a solvent, and for each of the three anion‐cation complexes investigated, the total energy value DE (intermolecular + solvation) of the associated ion pair is only slightly larger (≈ 5 kcal/mol out of ≈ 170) than that of the fully dissociated arrangement and has a virtually flat dependence as a function of intermolecular separation. The associated complex has an enhanced stability in DMSO, a trend accented in chloroform and carbon tetrachloride. In water, the acetic acid–imidazole complex is less than 1 kcal/mol more stable than the dissociated pair. Energy balances, taking into account the experimental values of protonation energies of acetate and imidazole, indicate that the acetate‐imidazolium complex is more stable than its nonzwitterionic counterpart, acetic acid‐imidazole in water. In carbon tetrachloride, by contrast, the two complexes are of similar stabilities in terms of internal energies. When we consider free instead of internal energies of solvation, the three organic solvents stabilize the complex between the neutral molecules. The investigation of the interaction of two methylguanidiniums in the polar solvents suggests that the solvation energy of a complex, larger than that of the two isolated entities, could be able to overcome their electrostatic repulsion. This results in a small preference in favor of the complex. Implications of the findings of this study, and future prospects of applications to molecular recognition and conformational studies of oligopeptides and possibly proteins, are discussed. © 1995 John Wiley &amp; Sons, Inc.

The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules.

The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.

Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp‐369 → Asn): A mechanism involving one zinc per active site

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution.

The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and in the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution. The enzyme exhibits a novel fold composed entirely of alpha-helices joined by connecting loops. The enzyme's most prominent structural feature is the arrangement of 10 core helices around a large central cavity. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, and are essential for enzymatic activity, were found on opposite walls of this cavity, with the aspartate side chains approximately 12 A apart and facing each other. The location and metal ion binding properties of these sequences suggest that the conserved aspartate residues participate in substrate binding of catalysis.

Targeting glucose oxidase at aspartate and glutamate residues with organic two-electron redox mediators.

The bimolecular rate constants for the reactions of five organic two-electron redox mediators with reduced glucose oxidase (GOx) were determined by measuring voltammetric electrocatalytic currents at glassy carbon electrodes in the presence of excess glucose under anaerobic conditions. The mediators studied were thionine, brilliant cresyl blue, azure A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M-1 s-1) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10(6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate residues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dopamine groups were obtained, with activities of 50% and 75%, respectively, relative to native GOx in a dye-peroxidase assay. Although the daunomycin derivative did not show measurable intramolecular electron-transfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH2 groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, the currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s-1) could be determined. This value is > 20 times larger than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling reagents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.