Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis

Site-specific mutagenesis was employed to study structure-functionrelationships at the substrate binding site of rat tissue kallikrein.Four kallikrein mutants, the Pro219 deletion (P219del), the34–38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34–38)IN],the Trp215Gly exchange (W215G) and the double mutant with Tyr99Hisand Trp215Gly exchange (Y99H:W215G) were created by site-directedmutagenesis to probe their function in substrate binding. Themutant proteins were expressed in Esclzerichia coli at highlevels and analyzed by Western blot. These mutant enzymes werepurified to apparent homogeneity. Each migrated as a singleband on SDS-PAGE, with slightly lower molecular mass (36 kDa)than that of the native enzyme, (38 kDa) because of their lackof glycosylation. The recombinant kallikreins are immunologicallyidentical to the native enzyme, displaying parallelism withthe native enzyme in a direct radioimmunoassay for rat tissuekallikrein. Kinetic analyses of K_m and k_cat using fluorogenicpeptide substrates support the hypothesis that the Tyr99–Trp215interaction is a major determinant for hydrophobic P2 specificity.The results suggest an important role for the 34–38 loopin hydrophobic P3 affinity and further show that Pro219 is essentialto substrate binding and efficient catalysis of tissue kallikrein.     

Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase

The aminopeptidase PepC is a cysteine peptidase isolated fromlactic acid bacteria. Its structural and enzymatic propertiesclosely resembles those of the bleomycin hydrolases, a groupof cytoplasmic enzymes isolated from eukaryotes. Previous biochemicaland structural data have shown that the C-terminal end of PepCpartially occupies the active site cleft. In this work the substratespecificity of PepC was engineered by deletion of the four C-terminalresidues. The mutant PepC432–435 cleaved peptide substratesas an oligopeptidase while the aminopeptidase specificity wastotally abolished. The substrate size dependency indicated thatPepC432–435 possesses an extended binding site able toaccommodate four residues of the substrate on both sides ofthe cleaved bond. The activity of PepC432–435 towardstryptic fragments of casein revealed a preference for peptideswith hydrophobic amino acids at positions P2 and P3 and forGly, Asn and Gln at position P1. PepC432–435 was shownto be highly sensitive to the thiol peptidase inhibitors leupeptinor E64 which are inefficient towards the wild-type PepC. Inconclusion, deletion of the four C-terminal residues in PepCproduces a new enzyme with properties resembling those of anendopeptidase from the papain family.     

Double-site ricin B chain mutants retain galactose binding

Three distinct double-site and two single-site ricin B chain(RTB) mutants were expressed in Spodoptera frugiperda insectcells and purified from infected cell supernatants. The yieldsof recombinant proteins were 0.01–0.2 mg/1. The purityafter monoclonal antibody affinity chromatography was 1–20%.The mutant proteins were soluble, immuno-reactive with monoclonalantibodies and polyclonal antibodies to RTB and demonstratedmolecular weights of 32 kDa, similar to plant RTB. All threedouble-site and both single-site mutants bound asialofetuinand mammalian cell surfaces based on an asialofetuin ELISA andcell binding immunofluorescence assay. While one double-sitemutant, W37S/Y248S, had a 1 log drop in sugar binding, the othertwo double-site mutants W37S/Y248H and D22E/D234E had 2 logreductions in sugar binding. Each mutant reassociated efficiently(25–75%) with plant ricin A chain (RTA) to form cytotoxicheterodimers. The concentration of protein required to reduceprotein synthesis 50% (ID₅₀) was 1 log higher than plant ricinfor W37S/Y248S-RTA and the single-site mutant heterodimers,Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin forthe other two double-site mutant heterodimers. The results suggestamino acid residues in both the 1 and 2 subdomains of RTB participatein sugar binding. However, other subdomains must contributeto the avidity of ricin for cell surface oligosaccharides.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans

The role of four aromatic residues (W85, Y172, W266 and W274)in the structure–function relationship in xylanase A fromStreptomyces lividans (XlnA) was investigated by site-directedmutagenesis where each residue was subjected to three substitutions(W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four aminoacids are highly conserved among family 10 xylanases and structuraldata have implicated them in substrate binding at the activesite. Far-UV circular dichroism spectroscopy was used to showthat the overall structure of XlnA was not affected by any ofthese mutations. High-performance liquid chromatographic analysisof the hydrolysis products of birchwood xylan and xylopentaoseshowed that mutation of these aromatic residues did not alterthe enzyme's mode of action. As expected, though, it did reducethe affinity of XlnA for birchwood xylan. A comparison of thekinetic parameters of different mutants at the same positiondemonstrated the importance of the aromatic nature of W85, Y172and W274 in substrate binding. Replacement of these residuesby a phenylalanine resulted in mutant proteins with a K_M closerto that of the wild-type protein in comparison with the othermutations analyzed. The kinetic analysis of the mutant proteinsat position W266 indicated that this amino acid is importantfor both substrate binding and efficient catalysis by XlnA.These studies also demonstrated the crucial role of these activesite aromatic residues for the thermal stability of XlnA.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

Engineering of the substrate-binding region of the sublilisin-like, cell-envelop proteinase of Lactococcus lactis

The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide _g1-casein(l–23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137–139AKTalong one side of the binding cleft produced the 137–139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137–139GLA mutant with increased activity andbroader specificity. Furthermore, the 137–139GDT mutanthad a specificity towards _g1,-casein(l–23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate     

Molecular recognition of the Lewis Y antigen by monoclonal antibodies

The murine monoclonal antibody BR55-2 is directed against thetumor-associated antigen Lewis Y oligosaccharide. The LewisY core antigen is a difucosylated structure consisting of fourhexose units. Analysis of binding profiles of lactoseries isomericstructures by BR55–2 suggest that the binding epitopeincludes the OH-4 and OH-3 groups of the ß-D-galactoseunit, the 6-CH₃ groups of the two fucose units and the N-acetylgroup of the subterminal ß-D-N-acetylglucosamine (ßDGlcNAc).To elucidate the molecular recognition properties of BR55–2for the Y antigen, BR55–2 was cloned, sequenced and itsthree-dimensional structure was examined by molecular modeling.The crystal structure of BR96, another anti-Lewis Y antibody,solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide,and the lectin IV protein in complex with a Lewis b tetrasaccharidecore were used as a guide to probe the molecular basis for BR55–2antigen recognition and specificity. Our modeling study showsthat BR55–2 shares similar recognition features for thedifucosylated type 2 lactoseries Lewis Y structure observedin the BR96-sugar complex. We observe that a major source ofspecificity for the Lewis Y structure by anti-Y antibodies emanatesfrom interaction with the ß-D-N-acetylglucosamineresidue and the nature of the structures extended at the reducingsite of the fucosylated lactosoamine.     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background

To change the substrate preference of carboxypeptidase Y theputative substrate binding pocket was subjected to random mutagenesis.Based upon the three-dimensional structure of a homologous enzymefrom wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216,Ile340 and Cys341 are the amino acid residues of carboxypeptidaseY that constitute S₁ the binding pocket for the penultimateamino acid side chain of the substrate. We developed a new andgenerally applicable mutagenesis strategy to facilitate efficientscreening of a large number of mutants with multiple changesin carboxypeptidase Y. The key feature is the elimination ofwild type background by introducing a nonsense codon at eachtarget site for subsequent mutagenesis by degenerate oligonucleotides.The entire hypothesized S₁ binding pocket and subsets of itwere subjected to saturation mutagenesis by this strategy, andscreening yielded a number of mutant enzymes which have up to150 times more activity (k_cat/K_m towards CBZ-LysLeu-OH thanthe wild type enzyme. All selected mutants with increased activityhave mutations at position 178. Mutagenesis of positions 215and 216 has virtually no effect on the activity, while mutatingpositions 340 and 341 generally reduces activity.     

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.     

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease Tl

Hydrophobic effects on binding of ribonuclease Tl to guaninebases of several ribonucleotides have been proved by mutatinga hydrophobic residue at the recognition site and by measuringthe effect on binding. Mutation of a hydrophobic surface residueto a more hydrophobic residue (Tyr45 – Trp) enhances thebinding to ribonucleotides, including mononucleotide inhibitorand product, and a synthetic substrate-analog trinudeotide aswell as the binding to dinucleotide substrates and RNA. Enhancementson binding to non-substrate ribonucleotides by the mutationhave been observed with free energy changes ranging from –2.2 to – 3 .9 kJ/mol. These changes are in good agreementwith that of substrate binding, –2.3 kJ/mol, which iscalculated from Michaelis constants obtained from kinetic studies.It is shown, by comparing the observed and calculated changesin binding free energy with differences in the observed transferfree energy changes of the amino acid side chains from organicsolvents to water, that the enhancement observed on guaninebinding comes from the difference in the hydrophobic effectsof the side chains of tyrosine and tryptophan. Furthermore,a linear relationship between nucleolytic activities and hydrophobicityof the residues (Ala, Phe, Tyr, Trp) at position 45 is observed.The mutation could not change substantially the base specificityof RNase Tl, which exhibits a prime requirement for guaninebases of substrates.     

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex

Molecular recognition and site-directed mutagenesis are usedin combination to identify kinetically, transition state interactionsbetween glucoamylase (GA) and the substrate maltose. Earlierstudies of mutant Glu180 – Gin GA had indicated a rolein substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly,P.J. and Svensson, B. (1990) Protein Engng, 3, 193–198).Here, changes in activation energies calculated from measuredk_cat/K_m values for a series of deoxygenated maltose analoguesindicate hydrogen bonding between the mutant enzyme and the3-OH group of the reducing end sugar ring. Using the same substrateanalogues and determining activation energies with wild-typeGA an additional hydrogen bond with the 2-OH group of maltoseis attributed to an interaction with the carboxylate Glu180.This novel combination of molecular recognition and site-directedmutagenesis enables an enzyme substrate transition state contactto be identified and characterized even without access to thethree dimensional structure of the enzyme. Given the distantstructural relationships between glucoamylases and several starchhydrolases (Svensson, B. (1988) FEBS Lett., 230, 72–76),such identified contacts may ultimately guide tailoring of theactivity of these related enzymes.     

Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions

Assumptions about the dependence of protein unfolding on theconcentration of urea have been examined by an extensive surveyof the equilibrium unfolding of barnase and many of its mutantsmeasured by urea denaturation and differential scanning calorimetry.The free energy of equilibrium unfolding and the activationenergy for the kinetics of unfolding of proteins are generallyassumed to change linearly with [urea]. A slight downward curvatureis detected, however, in plots of highly precise measurementsof logjtu versus [urea] (where k_u is the observed rate constantfor the unfolding of barnase). The data fit the equation logkk_u= logk_u^H₂O* + mk_u^*.[urea] – 0.014[urea]², where mk_u^*is a variable which depends on the mutation. The constant 0.014 was measured directly on four destabilized mutants and wildtype, and was also determined from a global analysis of data from>60 mutants of barnase. Any equivalent deviations from linearityin the equilibrium unfolding are small and in the same region,as determined from measurements on 166 mutants. The free energyof unfolding of barnase, G_U–F, appears significantly largerby 1.6 kcal mol^–1 when measured by calorimetry than whendetermined by urea denaturation. However, the changes in G_U–Fon mutation, G_U–F, determined by calorimetry and by ureadenaturation are identical. We show analytically how, hi general,the curvature in plots of activation or equilibrium energiesagainst [denaturant] should not affect the changes of thesevalues on mutation provided measurements are made over the sameconcentration ranges of denaturant and the curvature is independentof mutation.