Structural Determinants of Cold Adaptation and Stability in a Large Protein*

Salvino D'Amico, Charles Gerday, and Georges FellerDagger

From the Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium

Received for publication, March 28, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The heat-labile alpha -amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha -amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point Tm, the calorimetric enthalpy Delta Hcal, the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters kcat and Km. The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both kcat and Km by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha -amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cold-active and heat-labile alpha -amylase from the antarctic bacterium Pseudoalteromonas haloplanktis is an exceptional case because it is the largest known protein undergoing a reversible unfolding according to a two-state reaction pathway. It has been proposed that this unusual behavior is dictated by the requirement for an improved flexibility or plasticity of the protein molecule to perform catalysis at near-zero temperatures (1). Structural investigations have suggested that this is achieved by decreasing the number and strength of weak interactions stabilizing the native conformation (2-4). Accordingly, one can expect that any mutation designed to improve the stability of this fragile edifice will induce significant perturbations of the denaturation pattern, thereby giving evidence for the structural features linked to the unfolding parameters.

Small globular proteins that unfold according to a pure two-state process, i.e. reversibly and without a stable intermediate between the native and the unfolded states, have allowed researchers to establish the thermodynamic stability function accounting for the Gibbs energy change associated with their denaturation over physiological and nonphysiological temperature ranges. The current availability of this thermodynamic function, mainly gained by differential scanning calorimetry, represents much more than the simple physicochemical characterization of a polymer because it offers a powerful tool for investigation of the still unexplained "second genetic code," i.e. the driving force allowing a polypeptide to fold into a predetermined native and biologically active conformation (5). The validity of the bell-shaped stability curve, which predicts protein unfolding at both high and subzero temperatures, has been established by the experimental demonstration of cold unfolding (6). Proteins are said to be marginally stable because the positive Gibbs free energy change is only a small difference between the large enthalpic and entropic contributions in the temperature range restrained between the cold and hot melting temperatures. Basically, denaturation at high temperature is entropy-driven, because the large and positive thermal dissipative force exceeds the corresponding enthalpy change. By contrast, cold unfolding is enthalpy-driven and arises from the negative contribution of the hydration Gibbs energy of protein groups, mainly polar, at low temperatures (7).

Four parameters are usually assigned to the unfolding event recorded by differential scanning calorimetry, (i) the melting point of the unfolding transition; (ii) Delta Cp, the difference in heat capacity between the native and unfolded states arising from the transfer of buried groups in the protein to water, in the random coil; (iii) Delta Hcal, the calorimetric enthalpy corresponding to the total amount of heat absorbed during unfolding and recorded by the calorimeter; and (iv) Delta Heff, the effective or van't Hoff enthalpy calculated from the slope of the transition and describing the cooperativity of the unfolding reaction. In the case of a pure two-state model, Delta Hcal and Delta Heff are nearly identical. However, large proteins generally unfold irreversibly as a result of strong heat-induced aggregation, with large deviations from the two-state model because their multidomain structure induces a stepwise or overlapping unfolding of domains, behaving as individual calorimetric units (Fig. 1).

We report here the calorimetric characterization of mutants from the cold-active alpha -amylase defined on the basis of its strong homology with heat-stable mesophilic alpha -amylases. These mutants reveal the unsuspected contribution of single amino acid substitutions to the thermodynamic parameters characteristic of large heat-stable proteins and provide new insight into the molecular adaptations of enzymes from psychrophilic organisms.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenesis and Protein Purification-- Mutations in the P. haloplanktis alpha -amylase gene were introduced by polymerase chain reaction as described (8). The nucleotide sequence of the constructions was checked on an Amersham Pharmacia Biotech ALF DNA sequencer. The recombinant wild-type alpha -amylase and the mutant enzymes were expressed in Escherichia coli at 18 °C and purified by DEAE-agarose, Sephadex G-100, and Ultrogel AcA54 column chromatography, as described previously (9). Pig pancreatic alpha -amylase (PPA)1 was from Roche.

Differential Scanning Calorimetry (DSC)-- Measurements were performed using a MicroCal MCS-DSC instrument as detailed (1). Samples (~3 mg/ml) were dialyzed overnight against 30 mM MOPS, 50 mM NaCl, and 1 mM CaCl2, pH 7.2. Thermograms were analyzed according to a non-two-state model in which Tm, Delta Hcal, and Delta Heff of individual transitions are fitted independently using the MicroCal Origin software (version 2.9). The magnitude and source of the errors in the Tm and enthalpy values have been discussed elsewhere (10). Fitting standard errors on a series of three DSC measurements made under the same conditions in the present study were found to be ±0.05 K on Tm and ±1% on both enthalpies.

Thermal Inactivation-- Thermograms generated by starch hydrolysis were recorded on a MCS isothermal titration calorimeter. The technical background for such an experiment has been described elsewhere (11). Starch concentration was 2% in 50 mM Hepes, 50 mM NaCl, 1 mM CaCl2, pH 7.2, and was saturating during the time course of the experiment. Reactions were initiated by injecting 10-50 µl of a 30 µg/ml enzyme solution into the same buffer. The activity decay at 45 °C was recorded continuously for 30 min, and data were fitted on a monoexponential function to determine the first order constant rate of enzyme inactivation at 45 °C, S.E. <1%.

Enzyme Assay and Kinetics-- The kcat values were determined at 25 °C using 3.5 mM 4-nitrophenyl-alpha -D-maltoheptaoside-4,6-O-ethylidene as substrate (8). The kcat/Km values for substrate hydrolysis were determined from initial rates using the following relation. kcat/Km = v0/S0E0, which is valid at S0 Km for systems that obey Michaelis-Menten kinetics. The validity of this equation was ascertained by performing determinations at three substrate concentrations.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Selection of Potentially Stabilizing Mutations-- The psychrophilic alpha -amylase from P. haloplanktis (AHA) belongs to chloride-dependent alpha -amylases. This group includes all known animal alpha -amylases and the enzymes from some Gram-negative bacteria, covering a large range of living temperatures (12). The high degree of amino acid sequence identity between chloride-dependent alpha -amylases allowed us to construct a multiple sequence alignment of the available primary structures (12) and to detect about 20% of 453 residues that are specific to the cold-adapted enzyme. The involvement of these residues in the weak stability of the psychrophilic alpha -amylase was assessed by comparison of its crystal structure (3, 4) with that of PPA, the closest structural homolog, as well as with the structure of alpha -amylases from human salivary and pancreatic glands and from the insect Tenebrio molitor. Molecular modeling was then used to check the consistency of mutations aimed to introduce weak interactions found in mesophilic alpha -amylases in the heat-labile AHA. The selected mutations are listed in Table I along with the restored interactions. Following production in E. coli and purification, the stability and kinetic parameters of 14 mutant enzymes were determined (Table II). Mutants are listed according to their main properties, as discussed below. Fig. 1 depicts the microcalorimetric behavior of the recombinant wild-type AHA and of heat-stable alpha -amylases, including PPA taken as reference for mutation selection.

                              
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Table I
Selected mutations in this study

                              
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Table II
Stability and kinetic parameters of wild-type and mutant alpha -amylases


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Fig. 1.   Thermal unfolding of alpha -amylases recorded by DSC. The heat-stable PPA and BAA are characterized by higher Tm (top of the transition) and Delta Hcal (area under the transition) values, by a flattening of the transition and by the occurrence of calorimetric domains (deconvolutions are in thin lines). All thermograms are base line-subtracted and normalized for protein concentrations.

Enthalpic Stabilization by Electrostatic Interactions-- Enthalpic stabilization involves an increase of the denaturation enthalpy and of the melting point. Such stabilization of the heat-labile alpha -amylase is obtained when a salt bridge (N150D), polar aromatic interactions (V196F), or an additional H-bond (K300R) is introduced in the protein. Thermal unfolding of some of these mutants is illustrated in Fig. 2. These additional electrostatic interactions induce a slightly higher Tm value (Table II) associated, however, with a large increase of the calorimetric enthalpy (Delta Hcal). Characterization of the double mutant N150D/V196F revealed several interesting features. The effect of the mutations on the melting point is additive (Table II, Fig. 2). Moreover, both mutations affect Delta Hcal and Delta Heff differently, whereas their cumulative effect in the double mutant leads to a Delta Hcal/Delta Heff ratio at unity, indicative of a perfect two-state unfolding pattern.


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Fig. 2.   Enthalpic stabilization. Thermal unfolding of AHA and of single and double mutants (NV, N150D/V196F) characterized by increased Tm and Delta Hcal values.

Calorimetric Domain Acquirement by Nonpolar Groups-- Aliphatic side chains were introduced in AHA in order to reconstruct hydrophobic core clusters found in mesophilic alpha -amylases (Q164I, T232V, N288V, and M379V). Interestingly, each substitution results in a non-two-state unfolding of the mutant enzymes and in the appearance of two calorimetric units or domains (Fig. 3). As also shown in Fig. 3, the separation between both calorimetric units in the mutants can be improved by adjusting the experimental conditions, providing evidence for the coexistence of two protein domains of distinct stability. Deconvolution of the heat capacity function revealed that one domain always has an increased Tm compared with that of AHA (Table II), underlining the stabilization induced by the reconstituted hydrophobic cluster. By contrast, the lower Tm of the first domain seems to be the consequence of strain imposed in one domain that is not compensated in other protein regions. This is supported by the combination of mutations Q164I and V196F resulting in a mutant enzyme displaying two stabilized domains, as far as Tm is concerned. The mutant T232V deserves special comments. Indeed, the shape of its heat capacity function is similar to that of PPA (Fig. 3) as well as the relative contribution of each domain to Delta Hcal and Delta Heff (Table II). Therefore, this mutant consistently reproduces the microcalorimetric unfolding pattern of the heat-stable PPA but at lower temperatures.


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Fig. 3.   Calorimetric domains induced by additional nonpolar groups in hydrophobic core clusters (inset, T232V, pH 8.0).

Cooperativity of Unfolding-- The comparison of Delta Hcal and Delta Heff values allows us to analyze the unfolding cooperativity by microcalorimetry. Several mutations listed in Table II affect the unfolding cooperativity without noticeable change of Delta Hcal. Thermal unfolding of mutants Del6, L219R and R64E (Fig. 4) clearly illustrates the relation between Delta Heff and the steepness of the transition. Furthermore, the thermogram slope of mutants L219R and R64E is considerably decreased and approaches the profile of large and stable proteins such as Bacillus amyloliquefaciens alpha -amylase (BAA) (Fig. 1).


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Fig. 4.   Alteration of unfolding cooperativity. Decreasing the steepness of the transition maintains a higher level of native structures at elevated temperatures.

Folding and Unfolding Reversibility-- In these experiments, the mutant enzymes were 100% unfolded, then allowed to refold during a cooling period of 15 min at 15 °C. Reversibility was calculated by the recovery of Delta Hcal during a second scan (Table II). Substitution of a single amino acid side chain is sufficient to drastically alter this critical parameter of protein folding, as demonstrated by mutant N12R that displays only 25% renaturation. Mutants Q164I, V196F, and Q164I/V196F that are stabilized by hydrophobic groups also decrease the unfolding reversibility significantly. In the same conditions, unfolding of the heat-stable PPA and BAA is completely irreversible.

Thermal Inactivation Recorded by Isothermal Titration Calorimetry-- Determination of inactivation rate constants usually requires the recording of the residual enzyme activity after incubation at high temperature. In the case of AHA and of its mutants, the model of Lumry and Eyring (13) is valid:


<UP>N</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>U</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>I</UP> (Eq. 1)
where k2 is the first order rate constant of thermal inactivation. However, the various degrees of reversibility noted for the mutants (kinetically characterized by k-1) strongly impair the validity of the results obtained by conventional methods. We have therefore devised a new method using isothermal titration calorimetry and recording activity at 45 °C as the heat released by the hydrolysis of starch glycosidic bonds. This method provides a direct and continuous monitoring of activity at the denaturing temperature (Fig. 5). As a prerequisite, the same experiment was followed by fluorescence (without substrate) showing that the structural modifications accompanying a temperature shift from 15 to 45 °C are fast with a k1 value of about 0.04 s-1. Therefore, the transition N right-arrow U is one order of magnitude faster than the transition U right-arrow I, and the disappearance of the active state N can be taken as a measure of k2, as reported in Table II. It is significant that 10 of 14 mutants are protected against thermal inactivation and that mutants carrying newly introduced electrostatic interactions display the lowest inactivation rate constants (Fig. 5). A plot of unfolding reversibility versus thermal inactivation (Fig. 6A) shows that these parameters for the mutant enzymes are clustered between the respective values for AHA and PPA.


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Fig. 5.   Thermal inactivation recorded by isothermal titration calorimetry. Upper panel, following enzyme injection (arrow), activity at 45 °C is recorded as the heat flow of the reaction. Note the stability of PPA and the activity decay of AHA. Lower panel, semilogarithmic plot of residual activity versus time illustrating the lower inactivation rate of some mutants. NV, N150D/V196F.


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Fig. 6.   Correlation of stability (A) and kinetic (B) parameters for wild-type (open circle ) and mutant () alpha -amylases.

Kinetic Parameters-- The kinetic parameters kcat and Km were recorded using a chromogenic maltoheptaose oligosaccharide (Table II). From these results, four main aspects should be pointed out. (i) As reported previously, AHA possesses a higher activity (kcat) than PPA in order to compensate for the slow chemical reaction rates at low temperature. (ii) The general trend of the mutations is to decrease both kcat and Km. Fig. 6B shows that both kinetic parameters for mutants all fall within a narrow region of the plot, between the values of AHA and PPA. (iii) As a result, the mutations tend to raise the specificity constant or catalytic efficiency kcat/Km to values close to that of PPA (Table II). (iv) Most mutations protecting the enzymes against thermal inactivation also concomitantly decrease the kcat value.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stability of Large Proteins-- The magnitude of the contribution, as recorded by microcalorimetry, of a single amino acid side chain to the stability parameters of a large protein is one of the most significant features revealed by engineering the heat-labile alpha -amylase. Such individual contributions are generally masked in stable proteins by the complex network of interactions involved in the native state conformation and by the fact that frequently, heat-induced unfolding of large proteins is kinetically driven as a result of pronounced irreversibility. Our results show that the selected electrostatic interactions provide the largest contribution to stability per se, by increasing both Tm and Delta Hcal and by protecting mutants against thermal inactivation (Table II). Thermal unfolding of the double mutant N150D/V196F follows the two-state transition model and is essentially reversible. Accordingly, its thermodynamic stability can be analyzed using the modified Gibbs-Helmholtz equation:


&Dgr;G(T)=&Dgr;H<SUB>m</SUB>(1−T/T<SUB>m</SUB>)+&Dgr;Cp(T−T<SUB>m</SUB>)−T&Dgr;Cp <UP>ln</UP> (T/T<SUB>m</SUB>) (Eq. 2)
This function corresponds to the energy required to disrupt the native state (14) and is illustrated in Fig. 7. One can note that despite a modest increase of the melting point, the conformational energy of the double mutant is twice that of the wild-type AHA around 10 °C. Similarly, the contribution at 20 °C of the ion pair in N150D can be estimated to 4.4 kcal/mol, of the H-bond in K300R to 0.7 kcal/mol, and of a polar aromatic interaction in V196F (assuming two interplanar interactions) to 0.8 kcal/mol. These values are in good agreement with data obtained by ab initio calculations (15) or by experimental studies (16, 17). These free energy gains originate, however, from very large enthalpic-entropic compensation effects. The denaturation enthalpy and entropy can be calculated according to the relations (18):
&Dgr;H(T)=&Dgr;H<SUB>m</SUB>+&Dgr;Cp(T−T<SUB>m</SUB>) (Eq. 3)

&Dgr;S(T)=&Dgr;H<SUB>m</SUB>/T<SUB>m</SUB>+&Dgr;Cp <UP>ln</UP> (T/T<SUB>m</SUB>) (Eq. 4)
For instance, the denaturation enthalpy at 20 °C for AHA is 10.5 kcal/mol, and the entropic term (TDelta S) amounts to 2.2 kcal/mol. The corresponding values for mutant N150D are 60.9 and 48.2 kcal/mol, respectively. Therefore, the gain of 4.4 kcal/mol in conformational energy provided by the ion pair arises from a 6-fold increase of the denaturation enthalpy balanced by a 20-fold increase of the entropic cost. It should be added that mutant N150D restores a surface salt bridge. Although the net contribution of solvent-exposed ion pairs has been frequently questioned (19), our results demonstrate that such interactions can provide a substantial increment of conformational stability. This is especially relevant for thermophilic and hyperthermophilic proteins that are characterized by an abundance of surface ion pairs (20), in some instances organized in interconnected networks (21, 22). On the other hand, about 8 ion pairs, 15 arginine residues and 10 aromatic interactions are lacking in the crystal structure of AHA when compared with the heat-stable PPA (3). The disappearance of these electrostatic based features is obviously a main determinant for both the low Tm and melting enthalpy of the psychrophilic alpha -amylase.


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Fig. 7.   Stability curves of AHA and of the double mutant N150D/V196F (NV). Dashed line, theoretical curve for a heat-stable alpha -amylase, using data from Ref. 41.

It is generally agreed that the hydrophobic effect is the main factor in stabilizing folded conformations (23). As shown in Fig. 3 and Table II, the insertion of a nonpolar group into a hydrophobic core cluster creates a stabilization center in the protein and in the peculiar case of AHA promotes the appearance of calorimetric domains. Unfortunately, large deviations from the two-state model preclude any reliable calculation for these mutants. It should be noted, however, that the increases of Tm for the stabilized transition are of the same magnitude or even higher (M379V) than that provided by electrostatic interactions. Analysis of the hydrophobic clusters in AHA shows that 20 substitutions decrease the hydrophobicity index of the involved residues relative to PPA. As indicated by the calorimetric behavior of the mutations selected for this work (Fig. 3), the overall low core hydrophobicity of the heat-labile AHA accounts to a large extent for the lack of discrete unfolding intermediates. It is usually accepted that extreme cooperativity is only achieved in small molecules with tight packing of groups (14). This obviously is not the case for the large multidomain heat-labile alpha -amylase. As suggested by the mutation effects, its unfolding cooperativity arises from the very low number of interactions maintaining the native state, which therefore results in the simultaneous disruption of all structural elements and in the unusual two-state unfolding of this large protein. Considering protein adaptation to temperature, a practical implication of the flattening of the unfolding transition in thermostable alpha -amylases such as BAA (Fig. 1) is to maintain a higher level of native conformation at elevated temperatures. Fig. 4 shows that the same holds true between 45 and 55 °C for mutants of the heat-labile alpha -amylase with altered cooperativity. The relation between unfolding reversibility and thermal inactivation (Fig. 6A) is another unsuspected result gained from the alpha -amylase mutant analysis. Referring to Equation 1, mutants protected against thermal inactivation are also those that refold less efficiently, mimicking the behavior of thermostable alpha -amylases. This could be explained by the large number of all types of weak interactions required for proper folding and stability in mesophilic and thermophilic enzymes. As a consequence, there will be more possibilities of side reactions during refolding, either by intramolecular mismatch or by aggregation due to replacement of the regular intramolecular pairing by intermolecular interactions (17).

Cold Adaptation-- It was shown that all 24 side chains in the active site performing H-bonding to a large pseudosaccharide inhibitor in the transition state conformation are strictly conserved in both AHA and PPA crystal structures (4, 24). As a consequence, changes in primary structure that occur far from the active site should be responsible for the high specific activity of AHA. As a matter of fact, all mutations in this study designed to restore a mesophilic character are located far from the catalytic center and tend to decrease both kcat and Km (Fig. 6B). It has been convincingly argued that increases in flexibility of cold-adapted enzymes lead to a broader distribution of conformational states, translated into higher Km values for enzymes devoid of adaptive changes within the active site (25, 26). In this context, rigidifying the molecule or part of the molecule by the newly engineered weak interactions in the psychrophilic alpha -amylase should contribute to decreased Km values by reducing the conformational entropy of the binding-competent species. The kinetic parameters of the mutants (Fig. 6B) give experimental support to the previous hypothesis that temperature-adaptive increases in kcat occur concomitantly with increases in Km (26).

It has been proposed that stability and activity are not physically linked (27). Laboratory evolution can indeed yield nonnatural catalysts that are thermostable and active either at high (28) or low (29, 30) temperatures. Nevertheless, when the selection is only applied on low temperature activity (the relevant parameter for psychrophilic enzymes), especially by in vivo functional selection systems (allowing cell growth, for instance), the evolved enzymes generally display a concomitant reduced thermostability (31-34). It was assumed that the selective advantage for these optimized catalysts depends on a high kcat and not on maintaining stability (27). However, our structure-based approach demonstrates that natural mutations occurring in the cold-active alpha -amylase associate high activity and low stability. The convergence of the results from both random and rational studies stresses that the lack of selective pressure for stable proteins at low temperatures does not entirely account for the instability of psychrophilic proteins. On the contrary, heat lability or conformational plasticity seems to be intimately interlaced with sustained activity in cold environments. This is possibly the easiest route taken by natural selection on the evolutionary time scale or because large fluctuations around the native state are required by the energetics of substrate binding (35) and catalysis at low temperatures, an aspect that is not documented for such enzymes to date. The mutant N288V represents an extreme case. This mutant possesses a heat-labile domain with the lowest Tm (Table II) but is marginally stable even at 4 °C, with a half-life of 2 h at 25 °C and requiring fast purification procedures. This behavior is consistent with the proposal that AHA has reached a state close to the lowest accessible stability of its native state and cannot be further destabilized (1). If indeed stability is linked to activity in cold-adapted enzymes, this limitation explains the imperfect adaptation of psychrophilic enzymes, i.e. their activity at low temperatures remains lower than that of mesophiles at 37 °C.

Concluding Remarks-- This mutational analysis of a large protein demonstrates that the psychrophilic alpha -amylase has lost numerous weak interactions during evolution to reach the proper conformational flexibility at low temperatures. These adaptive adjustments contribute to improve kcat without alteration of the catalytic mechanism, because the active site architecture is not modified, but at the expense of a weaker substrate binding affinity. On the other hand, thermophilic enzymes strengthen the same type of interaction to gain in structural stability at high temperatures but at the expense of poor activity at room temperature (36-38). These aspects underline the continuum in the strategy of temperature adaptation in proteins and reinforce the concepts of "compromise" (39) between activity and stability leading to "corresponding states" (40) of enzymes adapted to different thermal environments.

    ACKNOWLEDGEMENTS

We thank N. Gérardin and R. Marchand for their skillful technical assistance.

    FOOTNOTES

* This work was supported by the European Community in the form of Network Contract CT970131, Biotech Program BIO4-CT96-0051, Région Wallonne-Direction Générale des Technologies Convention 1928, and Fonds National de la Recherche Scientifique Contract FRFC 2.4515.00.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium. Tel.: +32 4 366 33 43; Fax: +32 4 366 33 64; E-mail: gfeller@ulg.ac.be.

Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M102741200

    ABBREVIATIONS

The abbreviations used are: PPA, pig pancreatic alpha -amylase; AHA, P. haloplanktis alpha -amylase; BAA, B. amyloliquefaciens alpha -amylase; DSC, differential scanning calorimetry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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