Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation

The function of arginine 65, a conserved residue located atthe periphery of the active site cleft in yeast 3-phospho-glyceratekinase (PGK), has been investigated by site-directed mutagenesis.Mutant enzymes with glutamine, serine and alanine at position65 all have very similar kinetic properties. The maximum velocities,determined in the absence of sulfate anion, are - 100% higherthan the V_max of wild-type PGK. The K_m values are increased2- to 3-fold for ATP and 5- to 6-fold for 3-phosphoglycerate(3PG). These results demonstrate that arginine 65 is not essentialfor catalysis. In contrast to wild-type enzyme, the mutantsare not activated by sulfate ions. In addition, steady-statekinetic experiments indicate that the mutants are no longeractivated by high concentrations of either 3PG or ATP. The dissociationconstants for anions were determined by spectral titrationsof the R65Q mutant labeled with a chromophoric probe. The K_dfor 3PG is increased 6-fold, as compared to wild-type PGK, whereasthe K_d for ATP is essentially unchanged. The KA for sulfateis decreased < 2-fold. The suppression of substrate- andsulfate-dependent activation suggests that arginine 65 participatesin the regulatory mechanism responsible for activation of theenzyme.     

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusilus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency

Residue 75 on the flap, a beta hairpin loop that partially coversthe active site cleft, is tyrosine in most members of the asparticproteinase family. Site-directed mutagenesis was carried outto investigate the functional role of this residue in Rhizomucorpusilus pepsin, an aspartic proteinase with high milk-clottingactivity produced by the fungus Rhizomucor pusillus. A set ofmutated enzymes with replacement of the amino acid at position75 by 17 other amino acid residues except for His and Gly wasconstructed and their enzymatic properties were examined. Strongactivity, higher than that of the wild-type enzyme, was foundin the mutant with asparagine (Tyr75Asn), while weak but distinctactivity was observed in Tyr75Phe. All the other mutants showedmarkedly decreased or negligible activity, less than 1/1000of that of the wild-type enzyme. Kinetic analysis of Tyr75Asnusing a chromogenic synthetic oligopeptide as a substrate revealeda marked increase in k_cat with slight change in K_m, resultingin a 5.6-fold increase in k_cat/k_m. When differential absorptionspectra upon addition of pepstatin, a specific inhibitor foraspartic proteinase, were compared between the wild-type andmutant enzymes, the wild-type enzyme and Tyr75Asn, showing strongactivity, had spectra with absorption maxima at 280, 287 and293 nm, whereas the others, showing decreased or negligibleactivity, had spectra with only two maxima at 282 and 288 nm.This suggests a different mode of the inhibitor binding in thelatter mutants. These observations suggest a crucial role ofthe residue at position 75 in enhancing the catalytic efficiencythrough affecting the mode of substrate-binding in the asparticproteinases.     

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase

Site-directed mutagenesis has been used to explore the roleof two carboxylates in the active site of histidine decarboxylasefrom Lactobacillus 30a. The most striking observation is thatconversion of Glu197 to either Gln or Asp causes a major decreasein catalytic rate while enhancing substrate binding. This isconsistent with models based on X-ray diffraction results whichsuggest that the acid may protonate a reaction intermediateduring catalysis. The Asp197 protein undergoes a suicide reactionwith substrate, apparently triggered by inappropriate protonationof the intermediate. This leads to decarboxylation-dependenttransamination which converts the pyruvoyl cofactor to an alanine,inactivating the enzyme. Conversion of Glu66 to Gln affectsparameters of kinetic cooperativity. The mutation fixes theHill number at – 1.5, midway between the pH-dependentvalues of the wild-type enzyme.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by l,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH

The effect of the substitution of the active site histidine48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acidanalogue in porcine pancreas phospholipase A₂ (PLA₂) was studied.TAA was introduced biosynthetically using a his-auxotrophicEscherichia coli strain. To study solely the effect of the substitutionof the active site histidine, two nonessential histidines (i.e.His17 and His 115) were replaced by asparagines, resulting ina fully active mutant enzyme (His-PLA₂). In this His-PLA₂ thesingle histidine at position 48 was substituted by TAA withan incorporation efficiency of about 90%, giving a mixture ofHis-PLA₂ and TAA-PLA₂. Based on the charge difference at acidicpH, both forms could be separated by FPLC, allowing for thepurification of TAA-PLA₂ free from His-PLA₂. At pH 6, TAA-PLA₂has a fivefold reduced activity compared with His-PLA₂. Thisreduced activity paralells a reduced rate of covalent modificationwith p-nitrophenacyl bromide of TAA-PLA₂ compared with His-PLA₂.Competitive inhibition gave comparable IC₅₀ values for WT-PLA₂,His-PLA₂ and TAA-PLA₂. These results indicate that the reductionin activity is not caused by a different affinity for the substrate,but more likely results from a reduced k_cat value in TAA-PLA₂.The enzymatic activities for native and mutant PLA₂s were measuredat different pH values. For WT-PLA₂ and His-PLA₂ the activityis optimal at pH 6 and is strongly deminished at acidic pH,with no observable activity at pH 3. In contrast, TAA-PLA₂ isas active at pH 3 as at pH 6. Most likely, the decrease in activityobserved for WT-PLA₂ and His-PLA₂ is caused by the protonationof the active site His48, which is the general base involvedin the activation of the nucleophilic water molecule. In TAA-PLA₂,however, the active site residue TAA48 is unprotonated at bothpH 3 and 6 as a result of the low pK_a of TAA compared with histidine.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNA^Tyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expressionvector to construct the rat aldolase C expression plasmid, pRAC42.This plasmid produces active rat aldolase C in the transfectedE.coli host cells. The characteristics of the purified enzyme,e.g. mol. wt, electrophoretic mobilities and kinetic parameters,are indistinguishable from those of authentic rat brain aldolaseC. Three different tetrameric hybrid forms, C₃A, C₂A₂ and CA₃,in addition to C₄ and A₄, were found to be produced in the hostcell when E.coli was co-transfected with expression plasmidsfor rat aldolase C and for human aldolase A. Similarly, thehybrid forms, C₃B, C₂B₂ and CB₃, in addition to C₄ and B₄, werealso produced in the cells when co-transfected with the plasmidsfor rat aldolase C and for human aldolase B.     

Triple point mutation Asp10 -> His, Asn101 -> Asp, Argl48 -> Ser in T4 phage lysozyme leads to the molten globule

The triple amino acid replacement (Asp10 His, Asn101 Asp,Arg148 Ser) in T4 phage lysozyme was carried out by site-directedmutagenesis. At acid pH (2.7) the mutant is in a confonnationalstate with the properties of the molten globule: (i) the mutantprotein molecule is essentially compact; (ii) its CD spectrumin the near UV region is drastically reduced in intensity ascompared with the wild type protein spectrum; (iii) the CD spectrumin the far UV region indicates the presence of pronounced secondarystructure in the mutant; (iv) unlike the wild type protein themutant protein can bind the hydrophobic fluorescent probe, ANS.     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

Engineered disulfide bonds in recombinant human interferon-{gamma}: the impact of the N-terminal helix A and the AB-loop on protein stability

Insertion sites for cysteines with optimal stereochemistry forthe formation of unstrained disulfide bridges were identifiedin recombinant human interferon- (rhu-IFN-) by computer modelling.We have engineered two different disulfide cross-linked mutants,containing a pair of symmetry-related disulfide bonds, whichstabilize the N-termini of both monomers of the homodimenc protein.Mutations E7C and S69C allow the formation of an intramonomerdisuffide bond between helices A and D. In contrast, the A17Cand H111C mutations lead to a covalent cross-link between bothmonomers. The AB-loop is linked to helix F. The fluorescenceproperties of native and disulfide cross-linked proteins werestudied as a function of guanidine hydrochloride concentration.Melting temperatures (T_m) were calculated from the decreasein CD ellipticity at 220 nm. The induction of the antiviraleffect was measured using A549 fibroblast cells infected withencephalomyocarditis virus. The ability to induce the expressionof the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activatedcell scanning analysis. The stability of both mutants was stronglyenhanced against temperature- and cosolvent-induced unfolding.The T_m of mutant IFN- E7C/S69C was 15°C. All measured biologicalactivities of this mutant were equal to wild type. In the caseof the other mutant IFN- A17C/H111C, the T_m value was 25°C.This mutation abolishes nearly the entire biological activity(<1%) with no detectable changes of secondary structure inthe CD spectrum. Our results illustrate the importance of theN-terminal helix A and the AB-loop for the unfolding pathwayand thermodynamic stability of rhu-IFN-.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site

The consequences of active site mutations of the Escherichiacoli D-xylose isomerase (E.C. 5.3.1.5 [EC] ) on substrate bindingwere examined by fluorescence spectroscopy. Site-directed mutagenesisof conserved tryptophan residues in the E.coli enzyme (Trp49and Trpl88) reveals that fluorescence quenching of these residuesoccurs during the binding of xylose by the wild-type enzyme.The fluorescent properties of additional active site substitutionsat His101 were also examined. Substitutions of His101 whichinactivate the enzyme were shown to have altered spectral characteristics,which preclude detection of substrate binding. In the case ofH101S, a mutant protein with measurable isomerizing activity,substrate binding with novel fluorescent properties was observed,possibly the bound pyranose form of xylose under steady-stateconditions.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

Activation of E350A mutant maltodextrin phosphorylase by exogenously added acetate

Site-directed mutagenesis of E350 to alanine in Escherichiacoli maltodextrin phosphorylase reduced both enzyme activity(100-fold) and apparent binding of the oligosaccharide substrate(10-fold), suggesting a participation of this residue in bindingof the substrate in the ground and transition states. The E350Amutant enzyme was found to be activated up to 20-fold by exogenousacetate ions which substitute for the deleted side chain. Incontrast, apparent binding was not affected by acetate ions,indicating a dual role for the carboxylic group of this residuein catalysis and binding. Formate also appears to activate theE350A mutant enzyme, but this effect is obscured by the stronginhibitory effect of formate on the wild-type enzyme. For propionateions, a weak 2-fold activation was noticed, while other compoundslike trifluoroacetate and acetamide had no effects on the catalyticproperties of either the E350A mutant enzyme or wild-type enzyme.If E350 was substituted by a glutamine, no activation was observedupon the addition of acetate ions. However, a weak activationby formate was found, confirming that activation by acetateis caused by specific binding at the mutated site.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme frombacteriopbage T4 were used to explore the feasibility of usingsite-directed mutagenesis to generate isomorphous heavy-atomderivatives for protein crystallography. Cysteines 54 and 97,present in wild-type lysozyme, can be readily reacted with mercuricion to produce an excellent isomorphous heavy-atom derivative.Mutants with an additional cysteine at position 86,146,153 or157, or with Cys 97 replaced by Val, were engineered by site-directedmutagenesis. The mutant lysozyme Thr 157 - Cys reacts with mercuricchloride to give an excellent new derivatve although Cys 157is only -60% substituted with the heavy atom. The cysteine atposition 146 is largely buried but reacts readily with mercuricchloride. In this case the isomorphism is poor and the resultantderivative is of marginal quality. Cys 153 reacts rapidly withmercuric ion but the derivative crystals do not diffract. Themutant Pro 86 - Cys does not yield a particularly good heavy-atomderivative. This is due in part to a loss of isomorphism associatedwith the mutation. In addition, Cys 86 shows very little reactivitytowards mercurials even though it is fully exposed to solvent.The mutation Cys 97 Val was used to explore the possibilityof creating an independent derivative by deleting a heavy-atomsite already present in wild-type lysozyme. In all cases thatwere tested, the quality of the heavy-atom derivative was improvedby using as an isomorphous pair mercury-substituted mutant versusnon-substituted mutant rather than mercury-substituted mutantversus (non-substituted) wild-type lysozyme. Unexpectedly, thecysteines that are most exposed to solvent and most mobile areleast reactive toward mercuric chloride. The cysteines thatprovide the best heavy-atom sites are those that are locatedin surface crevices and are only partly exposed to solvent.     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease

Oligonucleotide-directed mutagenesis has been used to replaceglycine residues by alanine in neutral protease from Bacillussubtilis. One Gly to Ala substitution (G147A) was located ina helical region of the protein, while the other (G189A) wasin a loop. The effects of mutational substitutions on the functional,conformational and stability properties of the enzyme have beeninvestigated using enzymatic assays and spectroscopic measurements.Single substitutions of both G1y147 and Gly189 with Ala residuesaffect the enzyme kinetic properties using synthetic peptidesas substrates. When Gly replacements were concurrently introducedat both positions, the kinetic characteristics of the doublemutant were roughly intermediate between those of the two singlemutants, and similar to those of the wild-type protease. Bothmutants G147A and G189A were found to be more stable towardsirreversible thermal inactivation/unfolding than the wild-typespecies. Moreover, the stabilizing effect of the Gly to Alasubstitution was roughly additive in the double mutant G147A/G189A,which shows a 3.2°C increase in T_m with respect to the wild-typeprotein. These findings indicate that the Gly to Ala substitutioncan be used as a strategy to stabilize globular proteins. Thepossible mechanisms of protein stabilization are also discussed.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase {alpha}-subunits substituted at the same position

The surface properties of wild-type and six mutant -subunitsof tryptophan synthase substituted at the same position, 49,which is buried in the interior, were measured by surface tension,foaming and emulsifying properties to correlate the surfaceproperties with the stabilities. The conformational stabilitiesof the seven -subunits differed dramatically depending on thecharacteristics of the substituting residues [Yutani et al.(1987) Proc. Natl. Acad. Sci., 84, 4441–4444]. The mutantproteins substituted by isoleucine and phenylalanine in placeof glutamic acid at position 49 were more stable than the otherproteins and showed higher surface tension and lower foamingand emulsifying properties than the wild-type and other mutantproteins. Good correlations were observed between these surfaceproperties and values of the Gibbs free energy of unfoldingin water, of the proteins. This indicates that the surface propertiesof the -subunits of tryptophan synthase depend closely on theconformational stabilities.