Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteins.     

Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS

KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.     

How to improve nature: study of the electrostatic properties of the surface of alpha-lactalbumin

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.     

阳荷红色素的稳定性研究

对阳荷红色素分别在自然光、温度、pH值,Ca~(2+)、Zn~(2+)、K~+、Na~+、Fe~(3+)等金属离子存在条件下进行稳定性的研究。结果表明,酸性条件下阳荷红色素色泽较好,Ca~(2+)、Zn~(2+)对该色素的稳定性影响较小,K~+、Na~+、Fe~(3+)对阳荷红色素稳定性影响较大。研究为阳荷红色素提取过程中应注意的事项及其稳定性研究提供必要参考。     

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.     

Biophysical Studies of the Amyloid β‐Peptide: Interactions with Metal Ions and Small Molecules

Alzheimer's disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.     

Increasing the hydrophobic interaction between terminal W-motifs enhances the stability of Salmonella typhimurium sialidase. A general strategy for the stabilization of {beta}-propeller protein fold

Protein engineering of the ß-propeller protein aimedat enhancing the structural stability of the protein was carriedout using a monomeric single domain ß-propeller protein,Salmonella typhimurium sialidase, as a model. Ala53 and Ala69each located at strands B and C of the W1 motif were mutatedto Leu and Val, respectively, to increase the hydrophobic interactionbetween W1 and W6 motifs. The mutants showed enhanced stabilitytowards guanidine hydrochloride and thermal unfolding. Ala53Leushowed higher stability, probably owing to the capability ofthe mutated Leu to interact extensively with more residues involvedin the hydrophobic interactions between the terminal W-motifs.The mutations, which are located far from the active site, haveno significant effect on the enzymatic properties. The strategyto enhance the stability proposed here might be applied to theother ß-propeller proteins.     

Studies on enzymatic activity and conformational stability of muscle acylphosphatase mutated at conserved lysine residues

An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.      

Effects of sulfhydryl regents on the activity of lambda Ser/Thr phosphoprotein phosphatase and inhibition of the enzyme by zinc ion

Sulfhydryl reagents, such as dithiothreitol (DTT), affected the activity of Ser/Thr phosphoprotein phosphatases. Addition of DTT to the assay buffer increased the affinity of lambda Ser/Thr phosphoprotein phosphatase (lambda-PPase) for its Mn2+ cofactor. On the other hand, the enzyme was found to be inactivated simply by dilution in Tris buffer. The inactivation could be completely prevented by the presence of DTT or Mn2+ in the buffer. Further studies showed that oxidation or reduction of cysteine residues in lambda-PPase may not be the cause of the change in the enzyme activity. Without exception, mutation of all cysteine residues in lambda-PPase to serine did not convert the enzyme into a thiol-insensitive mutant. By careful examination of the effects of different sulfhydryl reagents, metal ion cofactors and substrates on lambda-PPase, it was found that the role of sulfhydryl reagents was the chelation of small amounts of inhibitory metal ions, which were present in plastic laboratory ware, such as disposable cuvets and tubes, with prevention of the enzyme from inactivation. One of the main contaminants found in plastic cuvets was Zn2+, which is a potent inhibitor of lambda-PPase. The inhibition of lambda-PPase by Zn2+ was characterized. Pre-treatment of the enzyme (1-4 nM) with 1 microM of ZnCl2 almost completely inhibited the enzymatic activity in response to 2 mM Mn2+. However, no significant inhibition was found when the enzyme was added to the assay mixture containing 1 microM Zn2+ and 2 mM Mn2+ . This confirms the sensitivity of the holoenzyme to inhibitory metal ions in vitro. The kinetic analysis indicated that the inhibitory metal ion might compete with Mn2+ to bind to the active site of lambda-PPase. This was further supported by the mutation of metal cofactor binding amino acid residues of the enzyme. Mutants which have less affinity for Mn2+ are also less sensitive to Zn2+. Our results suggest that inhibitory metal ions may induce a different structural conformation for lambda-PPase.      

Metal ions in neuroscience

Metal ions are believed to participate in many neurodegenerative conditions. In excitotoxic cell death there is convincing evidence for the participation of Ca(2+) and Zn(2+) ions although the exact molecular mechanisms by which these metals exert their effects are unclear. Only in one instance has the metal binding site of metalloenzymes been exploited for therapeutic purposes and this is the use of Li(+) in the treatment of bipolar affective disorder. Again the exact molecular target is not clear but is likely to involve a Mg(2+)-dependent enzyme of an intracellular signalling pathway. In Parkinson's disease, the selective loss of dopaminergic neurones in the substantia nigra may be caused by radical-mediated damage and there is good evidence to suggest that Fe(2+) or (3+) is important in promoting formation of radical species. The evidence that free radicals are important in mediating other neurodegenerative conditions is less strong but still substantial enough to suggest that removal of reactive oxygen species or preventing their formation may be a valid approach to therapy.     

The importance of peripheral sequences in determining the metal selectivity of an in vitro-selected Co(2+) -dependent DNAzyme

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.     

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions

The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.     

Comparison of different ranking methods in protein-ligand binding site prediction

In recent years, although many ligand-binding site prediction methods have been developed, there has still been a great demand to improve the prediction accuracy and compare different prediction algorithms to evaluate their performances. In this work, in order to improve the performance of the protein-ligand binding site prediction method presented in our former study, a comparison of different binding site ranking lists was studied. Four kinds of properties, i.e., pocket size, distance from the protein centroid, sequence conservation and the number of hydrophobic residues, have been chosen as the corresponding ranking criterion respectively. Our studies show that the sequence conservation information helps to rank the real pockets with the most successful accuracy compared to others. At the same time, the pocket size and the distance of binding site from the protein centroid are also found to be helpful. In addition, a multi-view ranking aggregation method, which combines the information among those four properties, was further applied in our study. The results show that a better performance can be achieved by the aggregation of the complementary properties in the prediction of ligand-binding sites.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.     

Probing the stabilizing role of C-terminal residues in trimethylamine dehydrogenase

In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotein, the C-terminal 17 residues of each subunit (residues 713- 729) embrace residues on the other subunit. The role of this unusual mode of interaction at the subunit interface was probed by isolating three mutant forms of trimethylamine dehydrogenase in which the C- terminus of the enzyme was deleted by five residues [delta(725-729], 10 residues [delta(720-729)] and 17 residues [delta(713-729)]. The solution properties and conformational states of the three mutant enzymes were investigated using optical, fluorescence and circular dichroism spectroscopies, ANS binding and a novel and conformationally sensitive hydrodynamic method. The data reveal that sequential deletion of the C-terminus of trimethylamine dehydrogenase does not affect significantly dimer stability or the overall structural integrity of the enzyme. However, deletion of the C-terminus severely compromises, but does not abolish, the ability of the enzyme to become covalently coupled with the redox cofactor FMN in the active site, located over 20 A from the C-terminus. Hydrodynamic studies reveal minor conformational changes in the deletion mutants that lead to a more compact enzyme structure. These conformational changes are probably transmitted to the active site via altering the interaction of the C-terminus with the second helix in the beta/alpha barrel of trimethylamine dehydrogenase, leading to poor flavinylation during the folding of the enzyme and assembly with FMN.      

Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met- Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.      

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.