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.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A₂ (PLA₂) islocated at the entrance to the active site. To study the roleof residue 31 in PLA₂, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA₂ for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 9–20 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveV_max values which range between 15 and 70% relative to the wild-typeenzyme. The V_max values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3–50times. The K_m values for the monomeric substrate and the k_mvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water.     

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.     

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

The ability to engineer proteins by directed evolution requiresfunctional expression of the target polypeptide in a recombinanthost suitable for construction and screening libraries of enzymevariants. Bacteria and yeast are preferred, but eukaryotic proteinsoften fail to express in active form in these cells. We haveattempted to resolve this problem by identifying mutations inthe target gene that facilitate its functional expression ina given recombinant host. Here we examined expression of HRPin Saccharomyces cerevisiae. Through three rounds of directedevolution by random point mutagenesis and screening, we obtaineda 40-fold increase in total HRP activity in the S.cerevisiaeculture supernatant compared with wild-type, as measured onABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)](260 units/l/OD₆₀₀). Genes from wild-type and two high-activityclones were expressed in Pichia pastoris, where the total ABTSactivity reached 600 units/l/OD₆₀₀ in shake flasks. The mutantsshow up to 5.4-fold higher specific activity towards ABTS and2.3-fold higher specific activity towards guaiacol.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues,Pro196, Pro197 and His199, that flank the catalytic nucleophile,Cys198. Pro196 and Pro197 have restricted mobility that couldbe important for the structural transitions known to be essentialfor activity. To test this hypothesis we obtained and characterized13 amino acid substitutions for Pro196, 14 for Pro197 and 14for His199. All of the Pro196 and Pro197 variants, except P197R,and four of the His199 variants complemented TS-deficient Escherichiacoli cells, indicating they had at least 1% of wild-type activity.For all His199 mutations, k_cat/K_m for substrate and cofactordecreased more than 40-fold, suggesting that the conserved hydrogenbond network co-ordinated by His199 is important for catalysis.Pro196 can be substituted with small hydrophilic residues withlittle loss in k_cat, but 15- to 23-fold increases in K_m^dUMP.Small hydrophobic substitutions for Pro197 were most active,and the most conservative mutant, P197A, had only a 5-fold lowerk_cat/K_m^dUMP than wild-type TS. Several Pro196 and Pro197 variantswere temperature sensitive. The small effects of Pro196 or Pro197mutations on enzyme kinetics suggest that the conformationalrestrictions encoded by the Pro–Pro sequence are largelymaintained when either member of the pair is mutated. Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.     

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.     

Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (k_cat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (K_M) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 – Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 – 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 – Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2.     

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent K_M for arachidonicacid and a minor(3-fold) change in its V_max. For linoleic acid,the variant has no change in its K_M and a 10-fold reductionin its V_max, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active siteof Escherichia coli alkaline phosphatase was investigated bysite-specific mutagenesis. A mutant version of alkaline phosphatasewas constructed with alanine in place of aspartic acid at position101. When kinetic measurements are carried out in the presenceof a phosphate acceptor, 1.0 M Tris, pH 8.0, both the k_cat andthe K_m, for the mutant enzyme increase by –2-fold, resultingin almost no change in the k_cat/K_m ratio. Under conditions ofno external phosphate acceptor and pH 8.0, both the k_cat andthe K_m for the mutant enzyme decrease by {small tilde}2-fold,again resulting in almost no change in the k_cat/K_m ratio. Thek_cat for the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-typeand mutant enzymes, as is the K₁ for inorganic phosphate. Thereplacement of aspartic acid 101 by alanine does have a significanteffect on the activity of the enzyme as a function of pH, especiallyin the presence of a phosphate acceptor. At pH 9.4 the mutantenzyme exhibits 3-fold higher activity than the wild-type. Themutant enzyme also exhibits a substantial decrease in thermalstability: it is half inactivated by treatment at 49°C for15 min compared to 71°C for the wild-type enzyme. The datareported here suggest that this amino acid substitution altersthe rates of steps after the formation of the phospho-enzymeintermediate. Analysis of the X-ray structure of the wild-typeenzyme indicates that the increase in catalytic rate of themutant enzyme in the presence of a phosphate acceptor may bedue to an increase in accessibility of the active site nearSerl02. The increased catalytic rate of this mutant enzyme maybe utilized to improve diagnostic tests that require alkalinephosphatase, and the reduced heat stability of the mutant enzymemay make it useful in recombinant DNA techniques that requirethe ability to heat-inactivate the enzyme after use.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

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.     

Protein engineering of cytochrome P450cam (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Mutations of the active site residues F87 and Y96 greatly enhancedthe activity of cytochrome P450_cam (CYP101) from Pseudomonasputida for the oxidation of the polycyclic aromatic hydrocarbonsphenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-typeP450_cam had low (<0.01 min^–1) activity with these substrates.Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, whilefluoranthene gave mainly 3-fluoranthol. Pyrene was oxidizedto 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with smallamounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrenegave 3-hydroxybenzo[a]pyrene as the major product. The NADHoxidation rate of the mutants with phenanthrene was as highas 374 min^–1, which was 31% of the camphor oxidation rateby wild-type P450_cam, and with fluoranthene the fastest ratewas 144 min^–1. The oxidation of phenanthrene and fluoranthenewere highly uncoupled, with highest couplings of 1.3 and 3.1%,respectively. The highest coupling efficiency for pyrene oxidationwas a reasonable 23%, but the NADH turnover rate was slow. Theproduct distributions varied significantly between mutants,suggesting that substrate binding orientations can be manipulatedby protein engineering, and that genetic variants of P450_cammay be useful for studying the oxidation of polycyclic aromatichydrocarbons by P450 enzymes.     

Making tissue-type plasminogen activator more fibrin specific

The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296–299or DDD 364–366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466–470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe k_cat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296–299, where the presenceof fibrin manifests significant increases in both k_cat and K_m.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity.     

Engineering the active center of the 6-phospho-{beta}-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-ß-galactosidasefrom Lactococcus lactis were replaced by the corresponding residuesin homologous enzymes of glycosidase family 1 with differentspecificities. Three mutants, W429A, K435V/Y437F and S428D/K435V/Y437F, were constructed. W429A was found to have an improvedspecificity for glucosides compared with the wild-type, consistentwith the theory that the amino acid at this position is relevantfor the distinction between galactosides and glucosides. Thek_cat/K_m for o-nitrophenyl-ß-D-glucose-6-phosphate is 8-foldhigher than for o-nitrophenyl-ß-D-galactose-6-phosphatewhich is the preferred substrate of the wild-type enzyme. Thissuggests that new hydrogen bonds are formed in the mutant betweenthe active site residues, presumably Gln19 or Trp421 and theC-4 hydroxyl group. The two other mutants with the exchangesin the phosphate-binding loop were tested for their abilityto bind phosphorylated substrates. The triple mutant is inactive.The double mutant has a dramatically decreased ability to bindo-nitrophenyl-ß-D-galactose-6-phosphate whereas the interactionwith o-nitrophenyl-ß-D-galactose is barely altered. Thisresult shows that the 6-phospho-ß-galactosidase and therelated cyanogenic ß-glucosidase from Trifolium repenshave different recognition mechanisms for substrates althoughthe structures of the active sites are highly conserved.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.     

Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain

Ricin A-chain (RTA) is an N-glycosidase which removes a specificadenine residue from the large rRNA of eukaryotic ribosomes.As a consequence, the ribosome is inactivated and protein synthesisis inhibited leading to cell death. This report describes theeffects on enzyme activity of specific mutations of the conservedactive site Glu177. The activity of mutant proteins was initiallyscreened using an in vitro translation system. It was foundthat mutagenesis of Glu177 to Lys led to an apparent total inactivationof the enzyme, Glu177 to Ala had a small effect on activity,whereas the conservative Glu177 to Asp mutation had a significanteffect. The properties of Glu177 to Asp were investigated moreclosely. Mutant protein was purified from an Escherichia coliexpression system and kinetic analysis of the depurination activityassessed using salt-washed yeast ribosomes. It was shown thatthe K, of the mutant protein was unchanged when compared todata of wild type RTA; however, the k_cat was significantly decreased(49-fold compared to wild type RTA). This suggests that Glu177plays a predominant role in the rate-limiting step of the enzymaticmechanism and not in substrate binding. These data are discussedin relation to other reports of ricin Glu177 substitutions.     

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.     

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.     

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.