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To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.

Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.

Cysteine is the only variant of D169, a cofactor-binding residue in thymidylate synthase, that shows in vivo activity. The 2.4 A crystal structure of Escherichia coli thymidylate synthase D169C in a complex with dUMP and the antifolate CB3717 shows it to be an asymmetric dimer, with only one active site covalently bonded to dUMP. At the active site with covalently bound substrate, C169 S gamma adopts the roles of both carboxyl oxygens of D169, making a 3.6 A S...H[bond]N hydrogen bond to 3-NH of CB3717 and a 3.4 A water-mediated hydrogen bond to H212. Analogous hydrogen bonds formed during the enzyme reaction are important for cofactor binding and are postulated to contribute to catalysis. The C169 side chain is likely to be ionized, making it a better hydrogen bond acceptor than a neutral sulfhydryl group. At the second active site, C169 S gamma makes a shorter (3 A) hydrogen bond to the 3-NH of CB3717, CB3717 is approximately 1.5 A out of its binding site and there is no covalent bond between dUMP and the catalytic cysteine. Changes to partitioning among productive and non-productive conformations of reaction intermediates may contribute as much, if not more, to the diminished activity of this mutant than reduced stabilization of transition states.

Enzyme stability can be an important parameter in the design of recombinant toxins because unstable proteins are often degraded before they can reach their cellular target. There is great interest in the design of human pancreatic ribonuclease variants that could be cytotoxic against tumoral cells. To this end, some residues in the protein need to be substituted, but this may result in a loss of stability. Previous papers have reported the production of N- and C-terminal human pancreatic ribonuclease variants with increased thermal stability. Here, we investigated the contribution of the different amino acid changes at the N-terminus of the protein to its thermostability increase. We show that this increase correlates with the helical propensity of the first alpha-helix of the protein. On the other hand, deletion of the four last residues of the protein does not affect its thermal stability. These results set the basis for the design of a human pancreatic ribonuclease template on which amino acid substitutions can be made that could render the enzyme cytotoxic, without an important loss in its stability.

A mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase (PfTIM) was created by mutating a tyrosine residue at position 74, at the subunit interface, to glycine. Tyr74 is a critical residue, forming a part of an aromatic cluster at the interface. The resultant mutant, Y74G, was found to have considerably reduced stability compared with the wild-type protein (TIMWT). The mutant was found to be much less stable to denaturing agents such as urea and guanidinium chloride. Fluorescence and circular dichroism studies revealed that the Y74G mutant and TIMWT have similar spectroscopic properties, suggestive of similar folded structures. Further, the Y74G mutant also exhibited a concentration-dependent loss of enzymatic activity over the range 0.1-10 microM. In contrast, the wild-type enzyme did not show a concentration dependence of activity in this range. Fluorescence quenching of intrinsic tryptophan emission was much more efficient in case of Y74G than TIMWT, suggestive of greater exposure of Trp11, which lies adjacent to the dimer interface. Analytical gel filtration studies revealed that in Y74G, monomeric and dimeric species are in dynamic equilibrium, with the former predominating at low protein concentration. Spectroscopic studies established that the monomeric form of the mutant is largely folded. Low concentrations of urea also drive the equilibrium towards the monomeric form. These studies suggest that the replacement of tyrosine with a small residue at the interface of triosephosphate isomerase weakens the subunit-subunit interactions, giving rise to structured, but enzymatically inactive, monomers at low protein concentration.

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degrees C which was 3.1 degrees C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.

A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.

An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis. Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195. By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all. These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation.

The various factors which contribute to protein stability have been extensively examined using mutant proteins, but the same kinds of substitutions have given different results depending on the substitution sites. Recently, the contributions of some stabilization factors have been quantitatively derived as parameters by a unique equation, considering the conformational changes due to the mutations using mutant human lysozymes [Funahashi et al. (1999) Protein ENG: 12, 841-850]. To evaluate these parameters estimated from the mutant human lysozymes, stability-structure datasets for the mutant T4 lysozymes were selected. The stabilities for the mutant T4 lysozymes could be roughly estimated using these parameters. Notable differences between the estimated and experimental stabilities were caused by the uncertainty in part of the structures due to some Arg and Lys residues fluctuating on the surface of the T4 lysozyme. Excluding these atoms from the estimation gave a good correlation between the estimated and experimental stabilities. These results suggest that the parameters of the various stabilization factors derived from the mutant human lysozymes are compatible with the mutant T4 lysozymes, although they should be improved with respect to some points using more information.

A computational model of the non-nucleoside inhibitor 8-Cl TIBO complexed with HIV-1 reverse transcriptase (RT) was constructed in order to determine the binding free energies. Using Monte Carlo simulations, both free energy perturbation and linear response calculations were carried out for the transformation of wild-type RT to two key mutants, Y181C and L100I. The newer linear response method estimates binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas. In addition, the change in stability of the protein between the folded and unfolded states was estimated for each of these mutations, which are known to emerge upon treatment with the inhibitor. Results from the calculations revealed that there is a large hydrophobic contribution to protein stability in the native, folded state. The calculated absolute free energies of binding from both the linear response, and also the more rigorous free energy perturbation method, gave excellent agreement with the experimental differences in activity. The success of the relatively rapid linear response method in predicting experimental activities holds promise for estimating the activity of the inhibitors not only against the wild-type RT, but also against key protein variants whose emergence undermines the efficacy of the drugs.

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C(delta) and atom Lys61-C(beta) (2.92 A) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.

To elucidate correlative relationships between structural change and thermodynamic stability in proteins, a series of mutant human lysozymes modified at two buried positions (Ile56 and Ile59) were examined. Their thermodynamic parameters of denaturation and crystal structures were studied by calorimetry and X-ray crystallography. The mutants at positions 56 and 59 exhibited different responses to a series of amino acid substitutions. The changes in stability due to substitutions showed a linear correlation with changes in hydrophobicity of substituted residues, having different slopes at each mutation site. However, the stability of each mutant was found to be represented by a unique equation involving physical properties calculated from mutant structures. By fitting present and previous stability data for mutant human lysozymes substituted at various positions to the equation, the magnitudes of the hydrophobicity of a carbon atom and the hydrophobicity of nitrogen and neutral oxygen atoms were found to be 0.178 and -0.013 kJ/mol.A(2), respectively. It was also found that the contribution of a hydrogen bond with a length of 3.0 A to protein stability was 5.1 kJ/mol and the entropy loss of newly introduction of a water molecules was 7.8 kJ/mol.

Two additional disulfide bonds and three combined thermostabilizing mutations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and combined to produce the double cysteine mutations N20C/ A27C and T72C/A471C. The double cysteine mutants were expressed efficiently in Saccharomyces cerevisiae, and disulfide bonds formed spontaneously after fermentation. At 50 degrees C, the single mutants N20C and A27C had decreased specific activity, whereas the specific activity of the double mutants N20C/A27C and T72C/A471C were similar to wild-type glucoamylase. The N20C/A27C mutation increased thermostability, with an increased activation free energy of 1.5 kJ/mol at 65 degrees C, while the single mutation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affect thermostability at pH 4.5. The N20C/A27C mutation was separately combined with two other thermostabilizing mutations, G137A and S436P. Thermostabilities of all of the combined mutated glucoamylases were additive. N20C/A27C/G137A glucoamylase had higher specific activity than wild-type glucoamylase from 45 to 67.5 degrees C. The disulfide bond between positions 20 and 27 connects the C-terminus of helix 1 and the following beta-turn, suggesting that this region is important for glucoamylase thermostability.

Free energy calculations were conducted to determine the relative stability of the unnatural amino acid mutants of T4 lysozyme norvaline (Nvl) and O-methyl-serine (Mse) and of alanine at residue 133, which is leucine in the native sequence. These calculations were performed both to assess the validity of the methodology and to gain a better understanding of the forces which contribute to protein stability. Peptides of different length were used to model the denatured state. Restraints were employed to force sampling of the side chain chi1 dihedral of the perturbed side chain, and the effect of protein repacking in response to mutation was studied through the use of different constraint sets. In addition, the convergence behavior and hysteresis of the simulations in the folded and unfolded states were determined. The calculated results agree well with experiment, + 1.84 versus + 1.56 kcal/mol for Mse-->Nvl and -3.48 versus -2.2 to -3.6 kcal/mol for Nvl-->Ala. We find that free energy calculations can provide useful insights to protein stability when conducted carefully on a well chosen system. Our results suggest that loss of packing interactions in the native state is a major source of destabilization for mutants which decrease the amount of buried nonpolar surface area and that subtle responses of the backbone affect the magnitude of the loss of stability. We show that the conformational freedom of the chi1 dihedral has a noticeable effect on protein stability and that the solvation of amino acid side chains is strongly influenced by interactions with the peptide backbone.

It was shown by Koshy et al. [1990, Proc. Natl Acad. Sci USA, 87, 8697-8701; 1994, Biochem. J., 299, 347-350] that the substitution of proline 30 by alanine (P30A) of Drosophila melanogaster and rat cytochromes c exhibited decreased stabilities in both the heme iron-methionine sulfur (Fe-S) bond and overall protein conformation. Now we have found that the stability properties of the equivalent mutant of Rhodobacter capsulatus cytochrome c2 (P35A) are somewhat different. Based on optical and NMR spectroscopies, the Rb.capsulatus P35A alkaline transition (pKalk) was found to be unchanged with respect to the wild type, suggesting that the mutation in Rb.capsulatus cytochrome c2 has little effect on the stability of the Fe-S bond. However, Rb.capsulatus conformational stability was found to be decreased by 1.6 kcal/mol in the oxidized state. The difference in the stability properties of the equivalent proline to alanine substitutions in various species underscores the importance of studying mutations in more than one species before drawing generalizations about the role of conserved residues in protein structure and function.

The structure of a thermostable Ala172Leu mutant, designated A172L, of 3-isopropylmalate dehydrogenase from Thermus thermophilus was determined. The crystal belongs to space group P2(1), with cell parameters a = 55.5 A, b = 88.1 A, c = 72.0 A and beta = 100.9 degrees. There is one dimer in each asymmetric unit. The final R factor is 17.8% with 69 water molecules at 2.35 A resolution. The mutation is located at the interface between domains and the C alpha trace of the mutant structure deviates from that of the native structure by as much as 1.7 A, while the structure of each domain barely changes. The mutant enzyme has a more closed conformation compared with the wild-type enzyme as a result of the replacement of Ala with Leu at residue 172. These structural variations were found independent of the crystal packing, because the structure of wild type was the same in crystals obtained in different precipitants. The hinge regions for the movement of domains are located around the active cleft of the enzyme, an observation that implies that the mobility of domains around the hinge is indispensable for the activity of the enzyme. The larger side chain at the mutated site contributed to the thermostability of the mutant protein by enhancing the local packing of side chains, and also by shifting the backbone of the opposing domain.

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.

The thermal stability of two single (K3R, K67R) and one double (K3R-K67R) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied to test Lys --> Arg substitution as an 'electrostatically conservative' strategy to increase protein stability. The K3R mutant displays an increased thermostability with respect to the wild-type enzyme, whilst a decreased stability was observed in the case of the K67R and K3R-K67R mutants. Concentration dependence of the apparent inactivation constant (kapp) of the latter mutants, as compared to that of the wild type enzyme and K3R mutant, indicates that their higher sensitivity to heat inactivation is due to a perturbation of the dimer association. These results are confirmed also by fluorescence anisotropy measurements of the internal probe Tyr149. The possible role of Arg67 in perturbing the dimer dissociation equilibrium toward the monomeric form is discussed.

A pathological variant of human phosphoglycerate kinase, phosphoglycerate kinase-Uppsala, associated with chronic nonspherocytic hemolytic anemia has been found to differ from the normal enzyme by substitution of an arginine at position 206 (corresponding to position 203 in yeast) by a proline. In order to understand the structural and functional consequences of this mutation, the corresponding mutant in yeast phosphoglycerate kinase was constructed. The three-dimensional structure of this mutant was resolved at 2.9 A. Although the overall structure is not modified, small local changes were observed. The kinetic parameters of the mutant were not found to be greatly affected, the catalytic constant being lowered by only 10-20%. The most significant difference when compared with the wild-type enzyme is a decrease in stability by about 3 kcal/mol. The physiological implications of this instability are discussed.

We have identified previously two critical positions for the thermostability of the highly thermostable alpha-amylase from Bacillus licheniformis. We have now introduced all 19 possible amino acid residues to these two positions, His133 and Ala209. The most favourable substitutions were to Ile and Val, respectively, which both increased the half-life of the enzyme at 80 degrees C by a factor of approximately 3. At both positions a stabilizing effect of hydrophobic residues was observed, although only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the gain in protein stability. The construction of double mutants showed a cumulative effect of the most favourable and/or deleterious substitutions. Computer modelling was used to generate a 3-D structure of the wild-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)8 barrel domain of alpha-amylase; Ala209 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix. The model suggests that replacement by, for example, a valine could fill this cavity and therefore increase intra- and interhelical compactness and hydrophobic interactions.

Site-directed mutagenesis was performed at Asp-Gly (48-49, 66-67, 101-102) and Asn-Gly (103-104) sequences of hen egg-white lysozyme to protect the enzyme against irreversible thermoinactivation. Because the lysozyme inactivation was caused by the accumulation of multiple chemical reactions, including the isomerization of the Asp-Gly sequence and the deamidation of Asn [Tomizawa et al. (1994) Biochemistry, 33, 13032-13037], the suppression of these reactions by the substitution of Gly to Ala, or the introduction of a sequence of human-type lysozyme, was attempted and the mutants (where each or all labile sequences were replaced) were prepared. The substitution resulted in the reversible destabilization from 1 to 2 kcal/mol per substitution. The destabilization was caused by the introduction of beta-carbon to the constrained position that had conformational angles within the allowed range for the Gly residue. Despite the decrease in the reversible conformational stability, the mutants had more resistance to irreversible inactivation at pH 4 and 100 degrees C. In particular, the rate of irreversible inactivation of the mutant, which was replaced at four chemically labile sequences, was the latest and corresponded to approximately 18 kcal/mol of the reversible conformational stability. Therefore, replacement of the chemically labile sequence was found to be more effective at protecting enzymes against irreversible thermoinactivation than at strengthening reversible conformational stability.

The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.

A number of mutations have been shown previously to stabilize T4 lysozyme. By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (delta delta G = 3.6 kcal/mol). This shows that it is possible to engineer a protein of enhanced thermostability by combining a series of rationally designed point mutations. It is also shown that this stabilization is achieved with only minor, localized changes in the structure of the protein. This is consistent with the observation that the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associated with the constituent single mutants. One of the seven substitutions, Asn116-->Asp, changes a residue that participates in substrate binding; not surprisingly, it causes a significant loss in activity. Ignoring this mutation, there is a gradual reduction in activity as successively more mutations are combined.

The 3-D structures of two chimeric enzymes (4M6T and 2T2M6T) between the Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases were analysed by X-ray diffraction in order to investigate their different thermostabilities. The structure of 2T2M6T was determined by the difference Fourier method and that of 4M6T by rigid body refinement, as based on the structure of the T. thermophilus enzyme. These structures were refined stereochemically to an R-factor of 0.193 at 2.5 A resolution for 4M6T and to an R-factor of 0.195 at 2.2 A resolution for 2T2M6T. The 3-D structures of 4M6T and 2T2M6T were very close to the structure of the T. thermophilus enzyme, conspicuous differences being at the molecular surface. In particular, 2T2M6T having a larger reduction in thermostability was more closely related to the T. thermophilus enzyme. However, their correlations between C alpha-atom displacements and the root squares of the temperature factors were significantly different from each other.

The effects of engineered disulfide bonds on autodigestion and thermostability of Bacillus subtilis neutral protease (NP-sub) were studied using site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the introduction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fully active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermostability, which, by using reducing agents, was shown to be caused by the introduction of the cysteines and not by the formation of the disulfides. Mutants containing one cysteine exhibited intermolecular disulfide formation at elevated temperatures, which, however, was shown not to be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of disulfide bridges is unsuitable for the stabilization of proteases. Possible explanations for this phenomenon are discussed.

Systematic replacement of the amino acid residues in Escherichia coli ribonuclease HI with those in the thermophilic counterpart has revealed that two mutations, His62-->Pro (H62P) and Lys95-->Gly (K95G), increased the thermostability of the protein. These single-site mutant proteins, together with the mutant proteins His62-->Ala (H62A), Lys95-->Asn (K95N) and Lys95-->Ala (K95A), were crystallized and their structures were determined at 1.8 A resolution. The crystal structures of these mutant proteins reveal that only the local structure around each mutation site is essential for the increase in thermostability. For each mutant protein, the stabilization mechanism is considered to be as follows: (i) H62P is stabilized because of a decrease in the entropy of the unfolded state, without a change in the native backbone structure; (ii) K95G is stabilized since the strain caused by the left-handed backbone structure in the typical 3:5 type loop is eliminated; and (iii) K95N is slightly stabilized by a hydrogen bond formed between the side-chain N delta-atom of the mutated aspargine residue and the main-chain carbonyl oxygen within the same residue.

The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.

Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.

The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0 degrees C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2 degrees C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.