Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties

Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in k_cat but to no changein K_m, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected K_m a more than k_cat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased k_catonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect K_m. The Trpl78-Aspmutation greatly decreased k_cat while increasing K_m, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.     

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.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat

The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of –170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the k_cat/k_m ratio were observed.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipaseA₂ in catalysis and substrate binding, using site-directed mutagenesis.A mutant was constructed containing Phe at position 69. Kineticcharacterization revealed that the Phe-69 mutant has retainedenzymatic activity on monomeric and micellar substrates, andthat the mutation has only minor effects on k_cat and K_m. Thisshows that Tyr-69 plays no role in the true catalytic eventsduring substrate hydrolysis. In contrast, the mutation has aprofound influence on the stereospecificity of the enzyme. Whereasthe wild-type phospholipase A₂ is only able to catalyse thedegradation of sn-3 phospholipids, the Phe-69 mutant hydrolysesboth the sn-3 isomers and, at a low (1–2%) rate, the sn-1isomers. Despite the fact that the stereospecificity of themutant phospholipase has been altered, Phe-69 phospholipasestill requires Ca²⁺ ions as a cofactor and also retains itsspecificity for the sn-2 ester bond. Our data suggest that inporcine pancreatic phospholipase A₂ the hydroxyl group of Tyr-69serves to fix and orient the phosphate group of phospholipidmonomers by hydrogen bonding. Because no such interaction canoccur between the Phe-69 side-chain and the phosphate moietyof the substrate monomer, the mutant enzyme loses part of itsstereospecificity but not its positional specificity.     

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate):D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13 [EC] ] was changedto arginine by site-directed mutagenesis. The k_cat of the resultingmutant protein, K146R, was 500 times slower than wild-type insteady-state kinetic assays for both cleavage and condensationof fructose-1,6-bis(phosphate), while the Km for this substratewas unchanged. Analysis of the rate of formation of catalyticintermediates showed K146R was significantly different fromthe wild-type enzyme and other enzymes mutated at this site.Single-turnover experiments using acid precipitation to trapthe Schiff base intermediate on the wild-type enzyme failedto show a build-up of this intermediate on K146R. However, K146Rretained the ability to form the Schiff base intermediate asshown by the significant amounts of Schiff base intermediatetrapped with NaBH₄. In the single-turnover experiments it appearedthat the Schiff base intermediate was converted to productsmore rapidly than it was produced. This suggested a maximalrate of Schiff base formation of 0.022 s^–1, which wasclose to the value of k_cat for this enzyme. This observationis strikingly different from the wild-type enzyme in which Schiffbase formation is >100 times faster than k_cat. For K146Rit appears that steps up to and including Schiff base formationare rate limiting for the catalytic reaction. The carbanionintermediate derived from either substrate or product, and theequilibrium concentrations of covalent enzyme-substrate intermediates,were much lower on K146R than on the wild-type enzyme. The greaterbulk of the guanidino moiety may destabilize the covalent enzyme-substrateintermediates, thereby slowing the rate of Schiff base formationsuch that it becomes rate limiting. The K146R mutant enzymeis significantly more active than other enzymes mutated at thissite, perhaps because it maintains a positively charged groupat an essential position in the active site or perhaps the Argfunctionally substitutes as a general acid/base catalyst inboth Schiff base formation and in subsequent abstraction ofthe C4-hydroxyl proton.     

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions

The role of electrostatic interactions between the ionizableAsp158 and the active site thiolate-imidazolium ion pair ofsome cysteine proteinases has been the subject of controversyfor some time. This study reports the expression of wild typeprocaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants fromEscherichia coli. Purification of autocatalytically maturedenzymes yielded sufficient fully active material for pH (k_cat/K_m)profiles to be obtained. Use of both uncharged and charged substratesallowed the effects of different reactive enzyme species tobe separated from the complications of electrostatic effectsbetween enzyme and substrate. At least three ionizations aredetectable in the acid limb of wild type caricain and the Gluand Asn mutants. Only two pK_a, values, however, are detectablein the acid limb using the Ala mutant. Comparison of pH activityprofiles shows that whilst an ionizable residue at position158 is not essential for the formation of the thiolate-imidazoliumion pair, it does form a substantial part of the electrostaticfield responsible for increased catalytic competence. Changingthe position of this ionizable group in any way reduces activity.Complete removal of the charged group reduces catalytic competenceeven further. This work indicates that hydronations distantto the active site are contributing to the electrostatic effectsleading to multiple active ionization states of the enzyme.     

Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage

We have demonstrated that an active enzyme can be expressedon the surface of a bacteriophage. The gene encoding alkalinephosphatase from Escherichia coli was cloned upstream of gene3, which encodes a minor coat protein of the filamentous bacteriophage,fd. A fusion protein of the correct size was detected from viralparticles by Western blotting. Ultrafiltration confirmed thatthe enzyme fusion behaves as part of a larger structure as wouldbe expected of an enzyme fused to a viral particle. Both wild-typealkaline phosphatase (Argl66) and an active site mutant (Ala166) expressed in this way retain catalytic activity and havequalitatively similar kinetic properties to free enzyme. Valueswere obtained for K_m of 72.7 and 1070 µM respectivelywhilst relative k_cat for the mutant was 36% of that for wild-type.Phage particles expressing alkaline phosphatase were bound toan immobilized inhibitor (arsenate-Sepharose) and eluted withproduct (20 mM inorganic phosphate). In this way, the functionalenzyme is co-purified with the DNA encoding it. This may permita novel approach to enzyme engineering based on affinity chromatographyof mutant enzymes expressed on the phage surface.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzymeat a low temperature was applied to isolate cold-adapted mutantsof a thermophilic enzyme. Four mutant enzymes with enhancedspecific activities (up to 4.1-fold at 40°C) at a moderatetemperature were isolated from randomly mutated Thermus thermophilus3-isopropylmalate dehydrogenase. Kinetic analysis revealed twotypes of cold-adapted mutants, i.e. k_cat-improved and K_m-improvedtypes. The k_cat-improved mutants showed less temperature-dependentcatalytic properties, resulting in improvement of k_cat (up to7.5-fold at 40°C) at lower temperatures with increased K_mvalues mainly for NAD. The K_m-improved enzyme showed higheraffinities toward the substrate and the coenzyme without significantchange in k_cat at the temperatures investigated (30–70°C).In k_cat-improved mutants, replacement of a residue was foundnear the binding pocket for the adenine portion of NAD. Twoof the mutants retained thermal stability indistinguishablefrom the wild-type enzyme. Extreme thermal stability of thethermophilic enzyme is not necessarily decreased to improvethe catalytic function at lower temperatures. The present strategyprovides a powerful tool for obtaining active mutant enzymesat lower temperatures. The results also indicate that it ispossible to obtain cold-adapted mutant enzymes with high thermalstability.     

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase

A mutant of Lactobacillus casei dihydrofolate reductase hasbeen constructed in which Thr63, a residue which interacts withthe 2'-phosphate group of the bound coenzyme, is replaced byalanine. This substitution does not affect k_cat, but producesan 800-fold increase in the K_m for NADPH, which reflects dissociationof NADPH from the enzyme-NADPH-tetrahydrofolate complex, anda 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociationconstant for the enzyme-NADPH complex. The difference in magnitudeof these effects indicates a small effect of the substitutionon the negative cooperativity between NADPH and tetrahydrofolate.Stopped-flow studies of the kinetics of NADPH binding show thatthe weaker binding arises predominantly from a decrease in theassociation rate constant. NMR spectroscopy was used to comparethe structures of the mutant and wild-type enzymes in solution,in their complexes with methotrexate and with methotrexate andNADPH. This showed that only minimal structural changes resultfrom the mutation; a total of 47 residues were monitored fromtheir resolved ¹H resonances, and of these nine in the binarycomplex and six in the ternary differed in chemical shift betweenmutant and wild-type enzyme. These affected residues are confinedto the immediate vicinity of residue 63. There is a substantialdifference in the ³¹P chemical shift of the 2'-phosphate ofthe bound coenzyme, reflecting the loss of the interaction withthe side chain of Thr63. The only changes in nuclear Overhausereffects (NOEs) observed were decreases in the intensity of NOEsbetween protons of the adenine ring of the bound coenzyme andthe nearby residues Leu62 and Ile102, showing that the substitutionof Thr63 does cause a change in the position or orientationof the adenine ring in its binding site.     

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain

A mutant of papain, where an inter-domain hydrogen bond betweenthe side chain hydroxyl group of a serine residue at position176 and the side chain carbonyl oxygen of a glutamine residueat position 19 has been removed by site-directed mutagenesis,has been produced and characterized kinetically. The mutationof Ser176 to an alanine has only a small effect on the kineticparameters, the k_cat/K_m for hydrolysis of CBZ-Phe-Arg-MCA bythe Serl76Ala enzyme being of 8.1 x 10⁴ /M/s compared with 1.2x 10⁵ /M/s for papain. Serine 176 is therefore not essentialfor the catalytic functioning of papain, even though this residueis conserved in all cysteine proteases sequenced. The pH-activityprofiles were shown to be narrower in the mutant enzyme by upto 1 pH unit at high ionic strength. This result is interpretedto indicate that replacing Ser 176 by an alanine destabilizesthe thiolate—imidazolium form of the catalytic site Cys25-Hisl59residues of papain. Possible explanations for that effect aregiven and the role of a serine residue at position 176 in papainis discussed.     

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.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

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.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

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.     

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS,EC 2.1.1.45) are hydrogen bond donors to the phosphate moietyof the substrate, dUMP. In order to investigate how these argininescontribute to enzyme function, we prepared complete replacementsets of mutants at each of the four sites in Lactobacillus caseiTS. Mutations of R23 increase K_m for dUMP 2–20-fold, increaseK_m for cofactor 8–40-fold and decrease k_cat 9–20-fold,reflecting the direct role of the R23 side chain in bindingand orienting the cofactor in ternary complexes of the enzyme.Mutations of R178 increase K_m for dUMP 40–2000-fold, increaseK_m for cofactor 3–20-fold and do not significantly affectk_cat. These results are consistent with the fact that this residueis an integral part of the dUMP-binding wall and contributesto the orientation and ordering of several other dUMP bindingresidues. Kinetic parameters for all R179 mutations except R179Pwere not significantly different from wild-type values, reflectingthe fact that this external arginine does not directly contactthe cofactor or other ligand-binding residues. R218 is essentialfor the structure of the catalytic site and all mutations ofthis arginine except R218K were inactive.     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.