Structural aspects for evolution of beta-lactamases from penicillin-binding proteins

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.     

Water-assisted reaction mechanism of monozinc beta-lactamases

Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism. This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available experimental data. The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.     

Mechanism and functional role of antibody catalysis

The light (L) chain of a model antibody (Ab) was deduced to contain a serine protease-like catalytic site capable of cleaving peptide bonds. The catalytic site is encoded by a germline V_L gene. The catalytic activity can potentially be improved by somatic sequence diversification and pairing of the L chain with the appropriate heavy chain. Autoimmune disease is associated with increased synthesis of antigen (Ag)-specific Abs, but the reasons for this phenomenon are not known. Only recently has attention turned to the functional role of the catalytic function. Preliminary studies confirm that the catalytic cleavage of peptide bonds is a more potent means to achieve Ag neutralization, compared to reversible Ag binding. Administration of a monoclonal Ab to VIP in experimental animals induces an inflammatory response in the airways, suggesting that catalytic autoantibodies to this peptide found in airway disease and lupus are capable of causing airway dysfunction. The phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalysts directed against tumor or microbial Ags by exposing the autoimmune repertoire to such Ags or their analogs capable of recruiting the germline V_L gene encoding the catalytic site.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: rescue by return.

As previously described (Pratt, R. F.; Hammar, N. J. J. Am. Chem. Soc. 1998, 120, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicyloyl cyclic phosphate) inactivates the class C beta-lactamase of Enterobacter cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recyclization reaction that regenerates salicyloyl cyclic phosphate rather than hydrolysis of the covalent intermediate. The inactivation, therefore, is a slowly reversible covalent modification of the active site. The thermodynamic dissociation constant of the inhibitor from the inactivated enzyme is 0.16 microM. Treatment of the inactivated enzyme with alkali does not produce salicylic acid but does, after subsequent acid hydrolysis, yield one molar equivalent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of acyclic acyl phosphates which transiently inactivate the P99 beta-lactamase by acylation (Li, N.; Pratt, R. F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Rather counterintuitively, in view of the relative susceptibility of phosphates and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K(i) = 10 microM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 x 10(3) s(-1) x M(-1) and 0.098 s(-1), respectively) were much slower than expected for noncovalent binding. This result strongly indicates that the inhibition reaction of the phosphonate also involves phosphylation of the active site. Hence, unlike the situation with bacterial DD-peptidases covalently inactivated by beta-lactams, the P99 beta-lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.     

DNA hydrolysis by monoclonal autoantibody BV 04-01

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg²⁺. Catalysis was asssociated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A₇C₇ATATAGCGCGT₂, as well as a preference for cleaving within CG-rich regions of dsDNA. Data nn specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg²⁺ coordination site composed of the L32Tyr and L27d His side chains and a DNA 3′-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V_H and V_L genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important—rarely examined—function for some antibody molecules.     

Enzymatic GTP hydrolysis: insights from an ab initio molecular dynamics study

Ab initio methods were used to shed light on fundamental aspects of the enzymatic mechanism of guanosine triphosphate hydrolysis in the Cdc42/Cdc42GAP complex. The calculations focused on the nucleophilic addition of the catalytic water molecule to the gamma-phosphate phosphorus atom. A large model system was required to correctly reproduce the electrostatic properties on the active site. The model turned out to reproduce most of the electrostatic field of the biological complex at the reactants. Our calculations established the H-bond pattern of the catalytic water (WAT), which turned out to interact with Q61 and T35, in the most stable conformation. This ruled out the possibility that the catalytic water transferred its proton directly to the gamma-phosphate. Furthermore, the calculations suggested that the electronic structure of WAT was very different from that in the bulk. Finally, this study showed that during the reaction, WAT transferred a proton to Gln61, consistent with the available X-ray data on a transition-state analogue/enzyme complex(19) and with the decrease of activity in the Q61E mutant.     

In vitro selection for catalytic activity with ribosome display

We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.     

Novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.     

Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover

Protein film voltammetry of NarGH catalysing nitrate reduction under steady state conditions provides information on events occurring within the enzyme during the catalytic cycle. In this discussion we have focused on exploring the ability of two simple catalytic schemes to reproduce the voltammetric response of NarGH; electron transfer to the enzyme's active site being described either by interfacial electron exchange (Scheme 1) or intramolecular electron delivery via the operation of an electron relay centre (Scheme 2). When the two electron reduced, catalytically competent active site of the enzyme is generated from the oxidised form in 'rapid', non-rate limiting steps of the catalytic cycle, the voltammetric behaviour of NarGH cannot be reproduced. Rather under all the conditions investigated, one electron reduction of the active site from a semi-reduced to a fully-reduced state is found to be crucial to progression through the enzyme's catalytic cycle. The catalytically relevant semi- and fully-reduced oxidation states of the NarGH active site are most likely to correspond to the Mo(V) and Mo(IV) states of the Mo(MGD)2 centre, respectively, although it is not possible to rule out the possibility that they correspond to molybdopterin based oxidation states as observed in other enzymes. We suggest that the rate of either conformational rearrangement within the semi-reduced active site or intramolecular electron delivery to the active site constitutes a defining feature in the catalytic cycle of NarGH and results in the napp approximately 1 appearance of the catalytic waveform.     

Nature of the catalytically labile oxygen at the active site of xanthine oxidase

In this paper we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized active site of xanthine oxidase at pH 6 and 10. These results indicate that the active site possesses one terminal oxygen ligand (Mo=O), two thiolate ligands (Mo-S), one terminal sulfido ligand (Mo=S), and one Mo-OH moiety. EXAFS analysis demonstrates that the Mo-OH bond shortens from 1.97 A at pH 6 to 1.75 A at pH 10, which is consistent with the generation of a Mo-O- moiety. This study provides convincing structural evidence that the catalytic oxygen donor at the oxidized active site of xanthine oxidase is Mo-OH rather than the Mo-OH2 ligation previously suggested by X-ray crystallography. These results support a mechanism initiated by base-assisted nucleophilic attack of the substrate by Mo-OH.     

Communication between the active site and the allosteric site in class A beta-lactamases

Bacterial production of beta-lactamases, which hydrolyze beta-lactam type antibiotics, is a common antibiotic resistance mechanism. Antibiotic resistance is a high priority intervention area and one strategy to overcome resistance is to administer antibiotics with beta-lactamase inhibitors in the treatment of infectious diseases. Unfortunately, beta-lactamases are evolving at a rapid pace with new inhibitor resistant mutants emerging every day, driving the design and development of novel beta-lactamase inhibitors. Here, we examined the inhibitor recognition mechanism of two common beta-lactamases using molecular dynamics simulations. Binding of beta-lactamase inhibitor protein (BLIP) caused changes in the flexibility of regions away from the binding site. One of these regions was the H10 helix, which was previously identified to form a lid over an allosteric inhibitor binding site. Closer examination of the H10 helix using sequence and structure comparisons with other beta-lactamases revealed the presence of a highly conserved Trp229 residue, which forms a stacking interaction with two conserved proline residues. Molecular dynamics simulations on the Trp229Ala mutants of TEM-1 and SHV-1 resulted in decreased stability in the apo form, possibly due to loss of the stacking interaction as a result of the mutation. The mutant TEM-1 beta-lactamase had higher H10 fluctuations in the presence of BLIP, higher affinity to BLIP and higher cross-correlations with BLIP. Our results suggest that the H10 helix and specifically W229 are important modulators of the allosteric communication between the active site and the allosteric site.     

Antibody synthesis induced by endogenous internal images

In this study, immunization with a vaccine consisting of multiple F(abt’)₂ fragments of affinity-purified antitetanus toxoid antibodies covalently bound to a carrier protein successfully induced antitetanus toxoid antibodies. Further studies showed that this vaccine preparation contained no biologically detectable tetanus antigen. The induced antitetanus antibody (Ab1t’) titer was higher than the titer of antibodies binding control antigens. The immunizing F(abt’)₂ preparation did not elicit a secondary antitetanus response from mice primed with tetanus toxoid and, hence, appeared free of tetanus epitopes. The specificity of Ab1t’ was established by absorption and inhibition with antigen. Immunization with antitetanus F(abt’)₂ (Ab1t’) fragments appears to have elicited naturally occurring autologous antitetanus toxoid antibody (Ab1t’) through an idiotypic pathway. As predicted by network theory, anti-idiotype (Ab2) and antitetanus (Ab1t’) cycled reciprocally. Clonotypic characterization of Ab1t’ using isoelectric focusing and affinity immunoblotting showed increases in Ab1t’ titer to be the result of increased synthesis by limited subsets of antitetanus toxoid B-cell clones and not increased synthesis by multiple clones, as is characteristic of antigen-driven Ab1 responses. Many Ab1 and Ab1t’ clonotypes had identical pIs, suggesting that they either share V region genes or are the product of the same B-cell clones. These findings indicate that immunization with polyclonal multivalent Ab1 preparations can trigger active synthesis of antibodies with the same specificity. The results provide further evidence for naturally occurring idiotypic cascades that could be exploited for studies of catalytic antibodies.     

Alkaline phosphatase catalysis is ultrasensitive to charge sequestered between the active site zinc ions

Escherichia coli alkaline phosphatase (AP) is a prototypical bimetalloenzyme, facilitating catalysis of phosphate monoester hydrolysis with two Zn2+ metal ions that are only 4 A apart. In the reaction's transition state, one of the nonbridging oxygen atoms of the transferred group appears to interact directly with the Zn2+ ion metallocluster. To determine the importance and the energetic properties of this interaction, we systematically varied the charge on this oxygen atom, exploiting the ability of AP to catalyze reactions of different classes of substrates. We observed that the AP catalytic proficiency correlates very well (R2 = 0.98) with the charge on this oxygen atom, over 8 orders of magnitude of catalytic proficiency. The slope of this linear correlation (31 +/- 2 kcal/mol per unit charge) is extraordinarily steep, indicating that AP greatly discriminates between differentially charged substrates. We suggest that this discrimination arises via an electrostatic interaction with the bimetallocluster. The dependence of the AP catalytic proficiency on the nonbridging oxygen charge is much larger than charge perturbation effects observed previously for other proteins. We propose that AP uses folding energy to position the two Zn2+ metal ions in close proximity, thereby creating an active site with a high electrostatic potential that is extraordinarily sensitive to the charge that "solvates" the metallocluster. The sensitivity of enzyme energetics to systematic variation in electrostatic properties provides a powerful measure of the active site environment. Future work comparing the sensitivity of related enzymes that have been optimized to catalyze different reactions will help reveal how natural selection has tuned related active sites to favor different reactions.     

The catalytic mechanism of an aspartic proteinase explored with neutron and X-ray diffraction

Hydrogen atoms play key roles in enzyme mechanism, but as this study shows, even high-quality X-ray data to a resolution of 1 A cannot directly visualize them. Neutron diffraction, however, can locate deuterium atoms even at resolutions around 2 A. Both neutron and X-ray diffraction data have been used to investigate the transition state of the aspartic proteinase endothiapepsin. The different techniques reveal a different part of the story, revealing the clearest picture yet of the catalytic mechanism by which the enzyme operates. Room temperature neutron and X-ray diffraction data were used in a newly developed joint refinement software package to visualize deuterium atoms within the active site of the enzyme when a gem-diol transition state analogue inhibitor is bound at the active site. These data were also used to estimate their individual occupancy, while analysis of the differences between the bond lengths of the catalytic aspartates was performed using atomic resolution X-ray data. The two methods are in agreement on the protonation state of the active site with a transition state analogue inhibitor bound confirming the catalytic mechanism at which the enzyme operates.     

Insights into the mechanism of N2O reduction by reductively activated N2O reductase from kinetics and spectroscopic studies of pH effects

Nitrous oxide reductase (N2OR) from Achromobacter cycloclastes (Ac) can be reductively activated with reduced methyl viologen over a broad range of pH. Activated Ac N2OR displays a complex activity profile as a function of the pH at which catalytic turnover is measured. Spectroscopic and steady-state kinetics data suggest that [H+] has multiple effects on both the activation and the catalytic reactions. These experimental results are in good agreement with previous theoretical studies, which suggested that the transition state is stabilized by H-bonding interactions between the active site and an N2O-derived intermediate bound to the catalytic CuZ cluster.     

argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli contains a dinuclear metalloactive site

The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).     

Electrochemical immunoassay platform for high sensitivity protein detection based on redox-modified carbon nanotube labels

We report a highly sensitive immunoassay protocol based on the use of redox-modified multi-walled carbon nanotubes (MWNTs) as electrochemical labels. The MWNTs were coated with methylene blue (MB) at an optically-determined loading of 3.41 × 10(-3) mol g(-1), and were then attached to secondary antibodies (Ab(2)) by adsorption. As a model analyte mouse IgG was collected by primary antibody (Ab(1))-coated magnetic beads. Following binding of the MB-MWNT-Ab(2) conjugates, IgG could be measured by MB reduction. Using differential pulse voltammetry for quantification, IgG was calibrated with a dynamic range of 0.1 pg mL(-1) to 100 pg mL(-1). Given the different possible Ab(1)-MB-MWNT-Ab(2) orientations on the magnetic beads, it was likely that not all the MB communicated with the electrode. A greater quantity of MB could be accessed by using the Fe(CN)(6)(3-/4-) redox couple as a solution phase mediator. This enabled us to lower the dynamic range down to 5 fg mL(-1) to 100 fg mL(-1).     

二维四角TiC单层片上的析氢反应研究

电解水是目前获得氢气的高效方法之一.过渡金属碳化物因其廉价且在析氢反应(HER)中表现出较高的催化活性而备受关注.我们利用第一性原理首先计算了新型二维四角TiC单层片的稳定性及电子性质,进而计算其表面不同活性位点、不同氢覆盖率下的吸附能、吉布斯自由能(△ GH*)等属性,并且将对应的微观结构进行了系统分析比较,同时结合...     

Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase

For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments: the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site Mo(V).     

Quantum chemical study on the coordination environment of the catalytic zinc ion in matrix metalloproteinases

X-ray analyses of matrix metalloproteinases (MMPs) have shown that the catalytic zinc ion (Zn1) can bind to one to three water molecules in addition to three conserved histidine residues. To estimate the relative stability of the possible Zn1 coordination structures in the active site of the MMPs, we carry out computational analyses on the coordination environment of the Zn1 ion in the gelatinase A enzyme (or matrix metalloproteinase 2; MMP-2). Four-, five-, and six-coordinated complexes representative of the Zn1 site are fully characterized by means of quantum mechanical (QM) methodologies. On one hand, B3LYP/LACVP* minimizations of various cluster models of the MMP-2 active site show that the trigonal bipyramidal geometry is energetically favored in the gas phase and that continuum solvent effects stabilize preferentially the tetrahedral complexes. On the other hand, B3LYP/OPLS-AA hybrid QM/molecular mechanical calculations in the solvated catalytic domain of the MMP-2 enzyme complemented with electrostatic Poisson-Boltzmann calculations show that the mature enzyme presents most likely a Zn1 ion coordinated by three histidine residues and two water molecules, while the active site glutamic acid is negatively charged. In consonance with X-ray diffraction data, other possible Zn1 configurations, a six-coordinated structure with Zn1-water as well as four- and five-coordinated complexes with a Zn1-bound hydroxide, are predicted to be very close in energy.