Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we "reverse engineered" PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.

Blocking PSD95-PDZ3's amyloidogenesis through point mutations that inhibit high-temperature reversible oligomerization (RO).

The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.

Thermodynamic Analysis of Point Mutations Inhibiting High-Temperature Reversible Oligomerization of PDZ3.

Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I6↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.

Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant.

Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.

Hydrophobic surface residues can stabilize a protein through improved water-protein interactions.

Protein stabilization is difficult to rationalize, but the detailed thermodynamic and structural analysis of a series of carefully designed mutants may provide experimental insights into the mechanisms underlying stabilization. Here, we report a systematic structural and thermodynamic analysis of bovine pancreatic trypsin inhibitor (BPTI) variants that are significantly stabilized through a single amino acid substitution at residue 38, which is located in a loop mostly exposed on the protein surface. Differential scanning calorimetry indicated that the BPTI-[5,55]Gly14 variants with a single mutation at position 38 were stabilized in an enthalpy-driven manner and that the magnitude of the stabilization increased as the hydrophobicity of residue 38 increased. This increase in the thermal stability of BPTI was unexpected because a hydrophobic residue on a protein surface is usually destabilizing. To identify the structural determinants of this stabilization, we determined the crystal structures of six BPTI-[5,55]Gly14 variants (Gly14 Gly38 , Gly14 Ala38 , Gly14 Val38 , Gly14 Leu38 , Gly14 Ile38 , and Gly14 Lys38 ) at high resolutions and showed that they retain essentially the same structure as the wild-type BPTI. A more detailed examination of their structures indicated that the extent of thermal stabilization correlated with both improved local packing and increased hydration around the substitution sites. In particular, the number of water molecules near residue 38 increased upon mutation to a hydrophobic residue suggesting that improved hydration contributed to the enthalpy-driven stabilization. Increasing a protein's thermal stability by the placement of a hydrophobic amino acid on the protein surface is a novel and unexpected phenomenon, and its exact nature is worth further examination, as it may provide a generic method for stabilizing proteins in an enthalpy-driven manner. DATABASE: The coordinates and structure factors of Gly14 Gly38 , Gly14 Ile38 , Gly14 Leu38 , and Gly14 Lys38 variants of BPTI-[5,55] are deposited in the Protein Data Bank under the PDB entry codes 5XX3, 5XX5, 5XX2, and 5XX4, respectively. We previously reported the structures of Gly14 Ala38 (2ZJX) and Gly14 Val38 (2ZVX).

Protocols of IATC, DSC, and PPC: The Multistate Structural Transition of Cytochrome c.

The recent development of high-precision calorimeters allows us to monitor the structural transition of biomolecules by calorimetry and thereby characterize the thermodynamic property changes accompanying three-dimensional structure changes. We developed isothermal acid-titration calorimetry (IATC) to evaluate the pH dependence of protein enthalpy. Using the double deconvolution method with precise differential scanning calorimetry (DSC), we revealed that the MG state is an equilibrium intermediate state of the reversible thermal three-state transition of the protein, and we successfully determined its volumetric properties by pressure perturbation calorimetry (PPC). Our findings underscore the importance of a precise calorimetry and analysis model for protein research.

Significance of H461 at subsite +1 in substrate binding and transglucosylation activity of amylomaltase from Corynebacterium glutamicum.

Amylomaltase (AM) catalyzes inter- and intra-molecular transglycosylation reactions of glucan to yield linear and cyclic oligosaccharide products. The functional roles of the conserved histidine at position 461 in the active site of AM from Corynebacterium glutamicum (CgAM) was investigated. H461 A/S/D/R/W were constructed, their catalytic properties were compared to the wild-type (WT). A significant decrease in transglucosylation activities was observed, especially in H461A mutant, while hydrolysis activity was barely affected. The transglucosylation factor of the H461A-CgAM was decreased by 8.6 folds. WT preferred maltotriose (G3) as substrate for disproportionation reaction, but all H461 mutants showed higher preference for maltose (G2). Using G3 substrate, kcat/Km values of H461 mutated CgAMs were 40-64 folds lower, while the Km values were twice higher than those of WT. All mutants could not produce large-ring cyclodextrin (LR-CD) product. The heat capacity profile indicated that WT had higher thermal stability than H461A. The X-ray structure of WT showed two H-bonds between H461 and heptasaccharide analog at subsite +1, while no such bonding was observed from the model structure of H461A. The importance of H461 on substrate binding with CgAM was evidenced. We are the first to mutate an active site histidine in AM to explore its function.

Thermodynamics of the Thermal Denaturation of Acid Molten Globule State of Cytochrome c Indicate a Reversible High-Temperature Oligomerization Process.

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ 1/2 I2 ⇄ 1/3 I3 ⇄ 1/4 I4) including three new oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible oligomerization.

Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD+. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H2M2) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the Km values for H2M2-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M4 and H4 whereas the Vmax values were similar; (2) the Km and Vmax values for H2M2-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H2M2 were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H2M2 was closer to that for M4 than for H4. We also show that HCQ had different affinities for various LDH isozymes.

Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum.

Amylomaltase catalyzes intermolecular and intramolecular transglucosylation reactions to form linear and cyclic oligosaccharides, respectively. The aim of this work is to investigate the structure-function relationship of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM). Site-directed mutagenesis was performed to substitute Tyr for Asn287 (N287Y) to determine its role in controlling amylomaltase activity and product formation. Expression of the wild-type (WT) and N287Y was achieved by cultivating recombinant cells in the medium containing lactose at 16 °C for 14 h. The purified mutated enzyme showed a significant decrease in all transglucosylation activities while hydrolysis activity was not changed. Optimum temperature and pH for disproportionation reaction were slightly changed upon mutation while those for cyclization reaction were not changed. Interestingly, N287Y showed a change in large-ring cyclodextrin (LR-CD) product profile in which the larger size was observed together with an increase in thermostability and substrate preference for G5 in addition to G3. The secondary structure of the mutated enzyme was slightly changed in related to the WT as evidenced from circular dichroism analysis. This work thus demonstrates that N287 is required for transglucosylation activities of CgAM. Having an aromatic residue in this position increased thermostability, changed product profile and substrate preference but demolished most enzyme activities.

Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.

Conserved and unique thermodynamic properties of lactate dehydrogenases in an ectothermic organism, the teleost Microstomus achne, and an endothermic organism, bovine.

It is widely believed that enzymatic activities in ectothermic organisms adapt to environmental temperatures. However, to date, no study has thoroughly compared multiple thermodynamic enzymatic characteristics across species living in dramatically different environments. To start to address this gap, we compared the characteristics of lactate dehydrogenase (LDH) purified from the muscles from slime flounder Microstomus achne white muscle and bovine skeletal muscle (bM4) and heart. The K m and V max for pyruvate reduction were about three times higher for M. achne LDH than bM4 Surprisingly, maximum LDH activity was observed at ∼30 °C and ∼50 °C for M. achne and bovine LDHs, respectively, suggesting that the maximum enzymatic activity of LDH is set at a temperature ∼20 °C higher than environmental or body temperature across species. Although K m and V max values of these LDHs increased with temperature, the V max/K m ratio for M. achne LDH and bM4 was independent. Differential scanning calorimetry and enthalpy change measurements confirmed that M. achne and bovine muscle-specific LDHs shared similar properties. Based on the present findings and previous reports, we hypothesize that the function and thermodynamic properties of muscle LDH are highly conserved between a teleost adapted to cold, M. achne, and bovine.

Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50 °C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40 °C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme.

IATC, DSC, and PPC Analysis of Reversible and Multistate Structural Transition of Cytochrome c.

Development of precise calorimeters has enabled us to monitor the structural transition of biomolecules by calorimetry to characterize the thermodynamic property changes accompanying three-dimensional structure change. We developed isothermal acid-titration calorimetry to evaluate the pH dependence of protein enthalpy, and demonstrated the thermodynamic transition between the native and molten globule (MG) states of cytochrome c with very small enthalpy change (~20 kJ/mol) by this method. The double deconvolution method with precise differential scanning calorimetry has revealed the MG state as an equilibrium intermediate state of the reversible thermal transition of the protein, and pressure perturbation calorimetry has succeeded in determining its volumetric properties. These examples strongly indicate the importance of a precise calorimetry and analysis model in the field of protein research.

The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.

BACKGROUND: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that ß-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular ß-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose. RESULTS: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked ß-1,4, ß-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7. CONCLUSION: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Unique structural changes in calcium-bound calmodulin upon interaction with protein 4.1R FERM domain: novel insights into the calcium-dependent regulation of 4.1R FERM domain binding to membrane proteins by calmodulin.

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca(2+) but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca(2+)-dependent manner. We have previously mapped a Ca(2+)-independent CaM-binding site, pep11 (A(264)KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca(2+) (Ca(2+)/CaM), interacts simultaneously with pep11 and with Ser(185) in A(181)KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca(2+)/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca(2+)/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca(2+). Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca(2+). Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca(2+)/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca(2+)/CaM-dependent regulation of R30 binding to membrane proteins.

Constant enthalpy change value during pyrophosphate hydrolysis within the physiological limits of NaCl.

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 M NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 M NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of -35 kJ/mol was obtained for the halophile and non-halophiles at 1.5-4.0 and 0.1-2.0 M NaCl, respectively. These results show that solvation changes caused by up to 4.0 M NaCl (water activity of ∼0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.