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Three Diverse Alcohol Dehydrogenases Remain Active at Salt Concentrations Greater than 1 M

Mehran Miroliaei*, Rasoul Sharifi, Peter J. Halling

Developmental and Molecular Biology Division, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

Abstract: A comparative study was carried out on the effects of a number of salts on enzyme activity of three representative alcohol dehydrogenases from non-halophilic sources. The enzymes from yeast (YADH), horse liver (HLADH) and Thermoanaerobacter brockii (TBADH) all retain significant activity at concentrations up to 4 M NaCl. In general catalytic activity follows the order NaCl > Na-acetate > Na₂SO₄ > NaNO₃ > NaClO₄. The deviation from the normal Hofmeister series may reflect effects in line with the “law of matching water affinity”, based on anion interactions with the Na⁺ cation in solution or the essential Zn²⁺ in the enzymes. Retention of activity generally follows the order YADH > HLADH > TBADH, which is opposite to the order of thermostability. Protein structural features promoting thermostability, like additional salt bridges, may lead to greater salt sensitivity. Comparison of cations Cs⁺, K⁺, NH⁴⁺ and Na⁺ showed weaker effects, not clearly in line with the Hofmeister series.

Keywords: alcohol dehydrogenase, hofmeister series, thermophilic enzyme, salts, non-halophilic protein

Received April 21, 2014; Revised May 07, 2014; Accepted May 07, 2014

Citation: Mehran Miroliaei, Rasoul Sharifi, Peter J. Halling, Three Diverse Alcohol Dehydrogenases Remain Active at Salt Concentrations Greater than 1 M. Biomedicine and Biotechnology, 2 (2). 37-41. January 2014. doi:10.12691/bb-2-2-2

1. Introduction

Enzymology is traditionally studied in dilute aqueous solution, with ionic strength up to 0.3 M or so. It is commonly believed that most enzymes will be inactive in concentrated salt solutions, and perhaps as a consequence this has been relatively little studied. There have of course been studies under these conditions of enzymes from halophiles, with discussion of how these proteins are adapted to be active at several molar salt concentrations [1,2,3,4]. In fact however, a number of “normal” enzymes have been shown to be quite active at such salt concentrations, even sometimes strongly activated compared with dilute solution [1,5-9]. A useful medium for enzymatic syntheses is mainly composed of solid particles, with a small aqueous phase between them in which the catalytic reaction takes place [10]. In such systems the aqueous phase is normally highly concentrated, and often this will include high levels of inorganic ions, for example when some reactants are added as salts. One enzyme applied practically in such systems is thermolysin, which is actually strongly activated by 4 M NaCl, as first reported in the 1970’s [11]. Enzyme activity at high salt concentrations is also of considerable practical importance in food systems [12]. There may also be messages for fundamental biology. In terms of the total concentrations of other solutes, cytoplasm could hardly be described as dilute aqueous. There has been something of a revival of interest recently on the effects of salt ions on proteins. This is particularly so for the specific effects dependent on the identity of the ions (“Hofmeister effects”), rather than simple electrostatic effects dependent on ionic strength [13,14,15]. These ion specific effects are believed to be related to properties like size, charge density, hydration energy, dispersion interactions, hydrophobicity and adsorption to non-polar surfaces. Effects on enzyme activity may reflect changes in secondary or tertiary structure, protein–protein interactions in multimeric enzymes, or interactions between substrates and the enzyme.

Alcohol dehydrogenases in general are useful biocatalysts for reduction of ketones and aldehydes, including the multimeric alcohol dehydrogenases (ADHs) from baker’s yeast (YADH), Thermoanaerobacter brockii (TBADH) and horse liver (HLADH) [16]. HLADH is a dimer with 375 residues, whereas both YADH and TBADH are tetrameric proteins, having 348 and 352 residues, respectively. While the yeast enzyme is somewhat unstable even at 25°C [17], the other two enzymes present high stability and activity at higher temperatures. Their importance for practical purposes makes them good candidates for enzyme activity assessment in media of high salt concentration. Thus a comparative study on the influence of a number of salts on the activity of these enzymes was carried out. The effects of high salt concentrations on the stability of YADH have been investigated by Ikegaya [18], although activity was not monitored in the high salt media. Alcohol dehydrogenases from halophilic archaebacteria have been shown to be highly activity in the presence of several molar salt concentrations [19,20]. We now report the activity of three representative alcohol dehydrogenases from non-halophilic sources in media with up to 4 M of a series of salts. Reasonable catalytic activity is retained even at the highest concentrations of NaCl.

2. Materials and Methods

2.1. Materials

Alcohol dehydrogenases from yeast (A7011), T. brockii, (A9287) and horse liver (expressed in E.coli, 55689), NAD⁺ (99% purity), NADP⁺ (95% purity), ethanol and 2-propanol were obtained from Sigma (). All salts were of analytical grade and obtained from Fisher scientific or Sigma-Aldrich.

2.2. Enzyme Assays

Enzyme activities were determined in a Beckmann-Coulter DU800 spectrophotometer equipped with temperature controller. YADH activity was determined at 25°C by monitoring rates of NAD reduction at 340 nm using the method described previously [5]. TBADH activity was measured at 65°C, following the reduction of 0.5 mM NADP+ (and monitoring the formation of NADPH) at 340 nm in the assay mixture containing 150 mM 2-propanol in 50 mM potassium phosphate, pH 7.8. Enzyme concentrations were determined by measuring absorbance at 280 nm and using the absorption coefﬁcients A1cm1%=12.6 for YADH [5] and =2.34×10⁴ M⁻¹cm⁻¹ for TBADH [17]. For HLADH activity measurement, the reduction of 0.5 mM NAD⁺ was followed in an assay mixture containing 0.17 M ethanol in 0.1 M sodium phosphate pH 8.6 at 37°C [21].

2.3. Kinetic Studies

Kinetic studies were done in the reaction mixture (1 mL) containing various concentrations of different salts with pH adjusted for optimal activity of each enzyme.

2.4. Preparation of Salt Solutions

Salt solutions were prepared by dissolving the required amount of each salt in sodium phosphate buffer at the assay pH. The apparent pH was then re-adjusted if necessary using NaOH or HCl to give the appropriate reading, with the electrode calibrated in the usual dilute standards. Salt solutions containing the alcohol substrate were then mixed with coenzyme and enzyme to start the reaction, in a spectrophotometer cell.

3. Results

Despite established ubiquitous Hofmeister effects on proteins and enzymes, no generalization has emerged from various studies since there are some examples showing that the salt-induced activity change does not follow the Hofmeister series [22,23,24]. A clear understanding of the underlying principles directing the mode of salt action would provide information useful for many practical purposes. The objective of the present investigation was to identify the impact of high salt concentrations (>1 M), where Hofmeister effects dominate, on the catalytic activity of three related alcohol dehydrogenases. The observed findings on catalytic activities are explained in terms of the combined differences in protein structure and the nature of inorganic salts utilized.

3.1. Effect of Salts on Yeast Alcohol Dehydrogenase

Figure 1 shows the effect of different sodium salts on the catalytic performance of the yeast enzyme (YADH). In the presence of 1 M Na₂SO₄, NaAc and NaCl, around half of original activity was preserved, while the addition of NaNO₃ further decreased the activity and NaClO₄ resulted in greater than 90% activity loss. Catalytic activity was less influenced by NaCl even at higher ionic strengths. Interestingly, no total loss of activity was observed even at 4 M concentration. The catalytic activity in the presence of monovalent anions follows the order: Cl- >CH₃COO⁻>SO₄2- >NO₃⁻ >ClO₄⁻ at 1-2 M and Cl- >CH₃COO- >NO₃- when salt concentration is 3-4 M. Note that sodium sulphate could not be used at more than 2 M, due to saturation of its solution.

Figure 1. Catalytic activity of yeast alcohol dehydrogenase in solution of sodium salts. Rates are presented relative to a control in the absence of added salt, with specific activity 71.6 µmol min⁻¹mg⁻¹

3.2. Effect of Salts on Horse Liver Alcohol Dehydrogenase

Figure 2. Catalytic activity of horse liver alcohol dehydrogenase in solution of sodium salts. Rates are presented relative to a control in the absence of added salt, with specific activity 45.8 µmol min⁻¹mg⁻¹

A trend similar to YADH was observed for the liver enzyme (HLADH), except for the unusually high retention of activity at 1 M of NaClO₄ (Figure 2). This same salt at 2 M or higher almost completely inactivated HLADH, as it did YADH. Once again activity was best retained in the presence of NaCl, with 10% of activity still observed even at 4 M. By and large, it appears that the catalytic activity of the liver enzyme is maintained rather less well in salt solutions than the yeast enzyme.

3.3. Effect of Salts on T. Brockii Alcohol Dehydrogenase

The influence of above salts on catalytic performance of TBADH was also studied (Figure 3). In this case the pattern of effects of the singly charged anions more clearly followed the Hofmeister series, with NaAc causing the least loss of activity, although Na₂SO₄ again gave relatively low activity. TBADH was completely inactivated in 1 M NaClO₄, thus being even more sensitive to this chaotropic anion than the other two enzymes. Moreover, in contrast to YADH and HLADH, higher concentrations of all tested salts were found to be very destabilising to the enzyme, with total inactivation at 2 M NaNO3 (Figure 3).

Figure 3. Catalytic performance of T. brockii alcohol dehydrogenase in solution of different sodium salts. Rates are presented relative to a control in the absence of added salt, with specific activity 40.0 µmol min⁻¹mg⁻¹

3.4. Effects of Different Cations (as Chlorides) on Yeast and Horse Liver Alcohol Dehydrogenase

Figure 4. Catalytic activity of yeast alcohol dehydrogenase in solution of chloride salts. Rates are presented relative to a control in the absence of added salt, with specific activity 71.6 µmol min⁻¹mg⁻¹

A comparative analysis of the influence of four different cations (Na+, NH₄+, Cs+, K+) was performed to determine their role in catalytic activity of the enzymes. The results are illustrated in Figure 4 and Figure 5. In general the effects of changing the cation are less than for anions, as commonly observed. Many of the differences are barely or not significant. With HLADH the retention of activity is noticeably higher with NaCl and NH₄Cl. Although a decrease of catalytic activity is evident for all studied salts, both enzymes remained active even at 3 M concentration. As in the experiments varying the anions, YADH generally retains activity better than does HLADH.

Figure 5. Catalytic activity of horse liver alcohol dehydrogenase in solution of chloride salts. Rates are presented relative to a control in the absence of added salt, with specific activity 45.8 µmol min⁻¹mg⁻¹

4. Discussion

Three ADH enzymes presented significantly different activities in solutions of inorganic salts. For most of the salts tested, the highest activities were retained with the yeast enzyme, while the lowest were found with the thermophilic enzyme, with intermediate effects on activity of the liver enzyme. Both YADH and HLADH retained significant activity even at 4 M concentrations of several salts, showing that these conditions are not as deleterious for catalytic activity of normal (non-halophile) enzymes as commonly supposed. Changes in the enzyme structure would be partly responsible for such inﬂuences. We have recently shown that HLADH is structurally more thermostable than YADH [25], while of course TBADH is known as very thermostable. So the higher the thermostability of the enzyme, the higher is the loss of catalytic activity in the presence of salts. This suggests that those factors determining thermal stability may be detrimental to activity retention in salt solutions. It is interesting that, even though YADH and HLADH are homologous enzymes from two mesophiles, HLADH is more similar to TBADH obtained from a hyperthermophile [25]. Higher thermal stability of TBADH is primarily due to the formation of an extended network of ion pairs and salt bridges (Lys257-Asp237-Arg304-Glu165) at the interface between subunits A and D (and B–C; [26]), mediated by water molecules. All four side-chain functional groups are located on the surface of the respective subunits [26,27]. It has been shown that TBADH contains nine conserved intrasubunit salt bridges, one of them (between Glu224 and Lys254) located in the coenzyme binding domain [28]. High salt concentrations may interact with these key charged groups, by shielding surface charges, promoting the binding of hydrated ions to the surface of protein, and changing the surrounding water structure. Indeed, examination of the crystal structure of TBADH [27] shows just such an ordered water molecule, in the small pocket located at a distance of 4.1Å from the sulfur atom of Cys-295 and 3.5Å from the β-carbon of Ile-86. Such ion pair networks do not exist in the mesophilic yeast and liver enzymes. Indeed, in HLADH two surface charged residues (Arg271 and Asp273), established as essential elements for enzymatic function, have been replaced by Ser and Ala respectively in YADH [29]. This may be one factor in the greater salt sensitivity of HLADH. It has also been suggested that more prominent hydrophobic interactions could contribute to the thermostability of thermophilic enzymes [30]. Hence, this may be another explanation for the higher inactivation of TBADH with NaClO4 and NaNO₃, whose chaotropic effects would disrupt these hydrophobic interactions, promoting exposure of buried side chains. A common feature observed in halophilic enzymes is an excess of surface negative charges, and this probably contributes to the higher salt tolerance we observe for YADH. Ratios of negative (Asp and Glu) to positive (Lys and Arg) charges can be calculated as 36/32 = 1.12 for YADH and 38/42 = 0.90 for HLADH [29]. Furthermore, high negative charge densities corresponding to the acidic residues at positions 226, 229, 230, 242, 246 and 248 in the coenzyme-binding domain of YADH are all replaced by nonpolar residues in HLADH [29]. A number of salt links in HLADH are disrupted in YADH: Arg-120 is deleted, Lys-168 replaced with Glu and Asp-323 and Lys-343 replaced with nonpolar residues [3]. Therefore, interference with the salt links may contribute to the higher sensitivity of HLADH to high salt concentrations. Greater salt resistance of YADH may originate from its intramolecular chaperone-like action, evolved by the presence of YSGVCHTDLHAWHGDWPLPVK [40-60]-sequence of the original structure [31]. It has been reported that high concentrations of NaCl strongly protect against dissociation of YADH, via quenching the electrostatic repulsion between Glu-101 of one subunit and Asp-236 of another subunit [32]. This may contribute to the observed higher salt stability of YADH.

The enzymes belong to the zinc-containing ADH family and their catalytic activity is absolutely dependent on maintenance of the integrity of zinc coordination at the catalytic site. The details of Zn coordination have been established for HLADH [33], YADH [34,35] and TBADH [36]. Because Zn²⁺ is a strongly kosmotropic cation it has a high tendency to bind with strongly kosmotropic anions, according to “the law of matching water afﬁnity” [37]. Although this metal ion incorporated in the enzyme's active site is coordinated, strongly kosmotropic anions may be powerful in competing with the normal coordinating ligands. This will significantly affect the functioning of the metal ion at the active site, so as to diminish the enzyme activity, account for the lower catalytic activity found in the presence of the most kosmotropic anions SO₄²⁻ and acetate. Particularly, strong interactions between this metal ion and the kosmotropic anions may result in a reduction in the nucleophilicity of the Zn²⁺-coordinated hydroxide ion, hence minimizing its nucleophilic attack. A similar mechanism was suggested for effects on alkaline phosphatase [7]. The greater loss of activity in the presence of NO₃⁻ and especially ClO₄⁻ is presumably a classic Hofmeister effect, which may reflect substantial changes in structure, or more local changes such as interactions with protein amino groups or effects on surface pH [38]. The mechanisms underlying the “law of matching water affinities” [37] may also contribute to the observed pattern of anion effects. Finally, in the case of SO₄²⁻, it should be noted that comparison with the other 1:1 salts is not so straightforward, since 1 M SO₄²⁻ is necessarily accompanied by 2 M Na⁺. As commonly observed, differences between the cations (Figure 4 and Figure 5) are less than between the anions (Figure 1- Figure 3). In addition, they do not clearly follow the expected Hofmeister series (Cs⁺ then K⁺/NH₄⁺ then Na⁺) in either the normal or reverse order. There are some similarities with trends observed for NADH oxidase [22].

5. Conclusion

All three enzymes retain substantial activity in the presence of high concentrations of various salts. The effects do not follow the full normal Hofmeister series, but instead the most kosmotropic anions give lower activities than Cl⁻. This may reflect effects in line with the “law of matching water affinities”. The activity reduction for three types of ADH follows the order: YADH<HLADH<TBADH, which is opposite to their thermostability order. This may reflect the structural adaptations of thermostable enzymes, such as increased salt bridges.

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