Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the {gamma}-L-glutamyl-L-cysteine-binding site

Lysl8, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathionesynthetase from Escherichia coli B are presumed to be highlyconcerned with the substrate, -L-glutamyl-L-cysteine (-Glu-Cys),binding by X-ray crystallography and affinity labeling studies.Using site-directed mutagenesis, we investigated functionalroles of those residues for -Glu-Cys binding. The mutant enzymesof Arg86 and Asn283 altered their kinetic parameters, especiallythe Michaelis constants of -Glu-Cys. In the case of Asn283,the residue is not likely to have an essential role in -Glu-Cysbinding but its side chain would extend to make a van der Waalscontact with bound -Glu-Cys. Chemical modification of a cysteineresidue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86would not only be much responsible for -Glu-Cys binding butwould also have a role in maintaining the structural integrityof the enzyme. The other mutant enzymes showed little defectin their kinetic parameters of -Glu-Cys.     

Redesigning the active site of Geotrichum candidum lipase

Attempts to engineer enzymes with unique catalytic propertieshave largely focused on altering the existing specificitiesby reshaping the substrate binding pockets. Few experimentshave aimed at modifying the configuration of the residues essentialfor catalysis. The difference in the topological location ofthe triad acids of Geotrichum candidum lipase (GCL) and thecatalytic domain of human pancreatic lipase (HPL), despite greatsimilarities in their topologies and 3-D structures, suggestthat these are related enzymes whose catalytic triads have beenrearranged in the course of evolution (Schrag et aL, 1992).In this study we prepared a double mutant GCL in which the catalytictriad acid is shifted to the position equivalent to the locationof the triad acid of HPL. The double mutant maintains 10% ofthe wild type activity against triglycerides and the fluorogenicester 4-methylumbelliferyl-oleate. The only significant differencesbetween the 3-D structures of the double mutant and wild typeGCL are at the mutated sites. Even the water structure in theregion of the triad is unchanged. The hydrogen bonding patternof the catalytic triad of the double mutant is very similarto that of pancreatic lipase. The acid of the double mutantis stabilized by only two hydrogen bonds, whereas three hydrogenbonds are observed in the wild type enzyme. These results stronglysupport the hypothesis that the pancreatic Upases are evolutionaryswitchpoints between the two observed arrangements of the catalytictriads supported by the /ß hydrolase fold and suggestthat this fold provides a stable protein core for engineeringenzymes with unique catalytic properties.     

Cytochrome P450 active site plasticity: attenuation of imidazole binding in cytochrome P450(cam) by an L244A mutation

We have identified a P450(cam) mutation, L244A, that mitigates the affinity for imidazole and substituted imidazoles while maintaining a high affinity for the natural substrate camphor. The P450(cam) L244A crystal structure solved in the absence of any ligand reveals that the I-helix is displaced inwards by over 1 A in response to the cavity created by the change from leucine to alanine. Furthermore, the crystal structures of imidazole-bound P450(cam) and the 1-methylimidazole-bound P450(cam) L244A mutant reveal that the ligands have distinct binding modes in the two proteins. Whereas in wild-type P450(cam) the imidazole coordinates to the iron in an orientation roughly perpendicular to the plane of the heme, in the L244A mutant the rearranged I helix, and specifically residue Val247, forces the imidazole into an orientation almost parallel to the heme that impairs its ability to coordinate to the heme iron. As a result, the imidazole is much more weakly bound to the mutant than it is to the wild-type enzyme. Despite the constriction of the active site by the mutation, previous work with the L244A mutant has shown that it oxidizes larger substrates than the wild-type enzyme. This paradoxical situation, in which a mutation that nominally increases the active site cavity appears to decrease it, suggests that the mutation actually increases the active site maleability, allowing it to better expand to oxidize larger substrates.     

Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or Asp119

In the cross-linking reaction of lysozyme between Leu¹²⁹ (-COO^–)and Lys¹³ (-NH₃⁺ using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC), a side reaction of the peptide bond inversionfrom to ß between A and Gly¹⁰² was greatly reducedby addition of ß-(1,4)-linked trimer of N-acetyl-D-glucosamine[(NAG)₃] When methylamine or 2-hydroxyethylamine was furtheradded, the extent of the cross-link formation was decreasedand the derivative where the -carboxyl group of Leu¹²⁹ was modifiedwith the amine was newly obtained. On the other hand, when ammoniawas added, the ß-carboxyl group of Asp¹¹⁹ insteadof the -carboxyl group was mainly amidated. From these results,the presence of a salt bridge between Asp¹¹⁹ and Arg¹²⁵ besidesthat between Lys¹³ and Leu¹²⁹ is proposed. Enzymatic activitiesof the derivatives prepared here indicated that the modificationof the -carboxyl group reduced the activity to {small tilde}90% of that of native lysozyme. Des-Leu¹²⁹ lysozyme, which lacksLeu¹²⁹ also showed {small tilde} 90% of the activity of nativelysozyme. Therefore, the salt bridge between Lys¹³ and Leu¹²⁹may play some role in maintaining the active conformation oflysozyine.     

Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity: mechanistic implication for amide hydrolysis by serine hydrolases

A lipase from Pseudomonas aeruginosa was subjected to directed evolution for increased amidase activity to probe the catalytic mechanism of serine hydrolases for the hydrolysis of amides. Random mutagenesis combined with saturation mutagenesis for all the amino acid residues at the substrate-binding site successfully identified the mutation at the residue 252 next to the catalytic H251 as a hot spot for selectively increasing the amidase activity of the lipase. The saturation mutagenesis targeted for the oxyanion hole (M16 and H83) gave no positive results. The substitutions of Met or Phe for Leu252 significantly increased the amidase activity toward N-(2-naphthyl)oleamide (2), whereas the esterase activity toward structurally similar 2-naphthyl oleate (1) was not affected by the substitution. The triple mutant F207S/A213D/M252F (Sat252) exhibited amidase activity (k(cat)/K(m)) 28-fold higher than that of the wild-type lipase. Kinetic analysis of Sat252 and its parental clone 10F12 revealed that the amidase activity was increased by the increase in the catalytic efficiency (k(cat)). The increase in k(cat) suggested the importance of the leaving group protonation by the catalytic His during the break down of the tetrahedral intermediate in the hydrolysis of amides.     

Analysis of several key active site residues of ricin A chain by mutagenesis and X-ray crystallography

Active she residues of ricin A chain were analyzed by sitedirectedmutagenesis and X-ray diffraction to help assess their rolesin the mechanism of action of this toxic N-glycosidase enzyme.Argl80 is thought, from X-ray studies, to protonate the adeninesubstrate at N3; this facilitates bond cleavage and is crucialto the mechanisms of action. The residue was converted to Glnand initial rate data measured. Km for the mutant is not significantlyaffected, increasing only 2-fold. The K_cat, however, is decreased 1000-fold. This is consistent with a simple interpretationthat Argl80 is involved more in transition state stabilizationthan in substrate binding. Tyrosines 80 and 123 are known fromX-ray models to stack on either side of the substrate adeninering. When they were each converted to serine overall activitywas reduced 160- and 70-fold respectively against ribosomesfrom Artemia salina. These effects are each 10 times greaterthan when the residues were previously converted to phenylalanines.Sufficient protein for the Tyr80 to Phe mutant was obtainedto carry out an X-ray analysis. Together with mutagenesis data,the structure suggests that the invariance of the two activesite Tyr residues is largely caused by structural stability.     

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activityof recombinant human tumor necrosis factor- (TNF) were investigatedby chemical modification and oligonucleotide-directed site-specificmutagenesis. TNF contains three histidine residues, locatedat positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate(DEP) was used to chemically modify TNF. The chemical inactivationof the in vitro cytotoxic activity of this lymphokine (usingmurine L929 target cells) was found to be time- and dose-dependent.Inactivated TNF failed to compete with fully bioactive [¹²⁵I]TNFfor human MCF-7 target cell receptors. Mutant polypeptides ofTNF were genetically engineered by oligonucoleotide-directedsite-specific mutagenesis. The cytotoxicity of a double histidinemutant, in which histidine-73 and histidine-78 were replacedwith glutamine, was not altered and was chemically inactivatedby DEP. Substituting glutamine for histidine-15 resulted in10–15% of the wild-type bioactivity. Replacing histidine-15with either asparagine, lysine or glycine resulted in a biologicallyinactive molecule. The data show that the histidine residueat position 15 is an amino acid that is required for the cytotoxicactivity of TNF.     

Restoration of pore-forming activity in staphylococcal {alpha}-hemolysin by targeted covalent modification

In previous studies, the replacement of His35 in the pore-formingprotein -hemolysin (HL) with Leu, lie, Pro, Arg, Ser, Thr orCys yielded inactive polypeptides. Here, we show that modificationof the inactive single-cysteine mutant HL-H35C with iodoacetamide,to form H35CamC, generates significant pore-forming activity.The closely related polypeptides H35N and H35Q have, respectively,essentially no activity and greatly reduced activity. The modifiedresidue in H35CamC, S-carboxamidomethylcysteine, mimicks histidinein volume, polarity and hydrogen bonding potential, but is unableto ionize. Unmodified H35C is defective in the final step ofpore formation: the conversion of an inactive heptameric membrane-boundassembly intermediate to a structure containing open channels.It is this step in assembly, that is ameliorated in H35CamC.     

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein

The 144 amino acid gene 5 protein of bacteriophage Pf1 bindstightly and cooperatively to single-stranded DNA during replicationof the phage genome. It has been suggested that aromatic aminoacid side chains are important for this interaction, probablythrough base stacking with the DNA. We have analysed the accessibilityof tyrosine residues in the DNA—protein complex, and theirimportance to the DNA-binding activity of the protein, by chemicalmodification and protection experiments using tetranitromethane.Tyrosines 21, 30 and 55 are surface accessible in the free proteinbut are protected from modification in the complex with phageDNA. Moreover, modification of these residues in the free proteinabolishes the ability to bind to DNA or oligonucleotides, asjudged by fluorescence spectroscopy and gel retardation analysis.Modification of the protein also results in the formation ofan intersubunit covalent cross-link between Tyr55 and Phe76,suggesting that Phe76 is located within the DNA-binding cleftof the protein. It is proposed that residues 17–34 ofthe Pf1 gene 5 protein form a beta-hairpin analogous to the‘DNA-binding wing’ of the fd and Ike gene 5 proteins.We suggest the existence of a single-stranded DNA binding motif,in which Tyr30 of the Pf1 protein is equivalent to the functionallyimportant Tyr26 of the fd gene 5 protein.     

The active site of carboxypeptidase Taq possesses the active-site motif His-Glu-X-X-His of zinc-dependent endopeptidases and aminopeptidases

Carboxypeptidase (CPase) Taq possesses the His–Glu–X–X–Hissequence, which is the consensus sequence in the active siteof zinc-dependent endopeptidases and amino-peptidases, at positions276–280. Amino acid replacement of the conserved His andGlu drastically diminished the activity of CPase Taq, and thezinc content of the enzyme was also greatly reduced when eitherof the two His residues was replaced with Arg or Tyr. The resultsindicate that this sequence actually functions as the activesite in CPase Taq, showing that CPase Taq is a novel type ofzinc-dependent CPase that possesses the His–Glu–X–X–Hisactive-site motif.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Novel features of serine protease active sites and specificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases and epidermolytic toxins

With a view to obtaining a better understanding of the structuraldeterminants of P1 glutamate specificity in glutamate-specincendopeptidases (GSEs), the active sites and specificity pocketsof such enzymes from Bacillus ticheniformis (gse-bl), Bacillussubtilis (mpr) and Staphylococcus aureus (v8 protease) weremodelled. This approach was extended to the epidermolytic toxins(ETs), responsible for the staphylococcal scalded skin syndrome.We identify a canonical structure for the S1 subsite, composedof H213 and T190, both of which we predict to interact directlywith the P1 glutamate. The possible importance of R30 (for gse-bland mpr) and of the N-terminus (for gse-bl, mpr and v8 protease)was also examined. In the case of mpr, a G193C substitutionis predicted to participate in a novel disulphide bridge whichstabilizes C193 in such a way as to maintain the oxyanion hole.In v8, the loss or substitution of several important structuralcomponents around D102 of the catalytic triad probably explainsits reduced catalytic efficiency in comparison with other GSEs.In the case of the epidermolytic toxins K216 may be importantfor the previously reported phospholipase C-like activity, sincethe model predicts that it may stabilize the negative chargeon the phosphonyl group.     

Activity of linked HIV-1 proteinase dimers containing mutations in the active site region

Mutations were introduced into the active site triplet (Asp–Thr–Gly)of one or both subunits of a linked dimer of human immunodeficiencyvirus type 1 proteinase. Mutation of Thr to Ser in one or bothsubunits did not alter the activity of the enzyme substantially,whereas its mutation to Asn in one subunit caused a dramaticdecrease in catalytic efficiency. Mutation of Gly to Val inone subunit also yielded an enzyme with very low activity. Theenzymes containing Thr Asn and Gly Val mutations in both subunitsresulted in inactive enzymes, based on their inability to self-processand on assay with an oligopeptide substrate. The dramatic decreasein enzyme efficiency of the mutants was interpreted using molecularmodels of the enzymes.     

Increased thermal and organic solvent tolerance of modified horseradish peroxidase

Horseradish peroxidase (HRP) was modified by maleic anhydride and citraconic anhydride. The thermal and organic solvent tolerances of native and modified enzyme were compared. These chemical modifications of HRP increased their thermostability both in aqueous buffer and some organic solvents, and also enhanced their tolerances of some organic solvents. We have studied the unfolding of native and modified HRP by heat to determine the conformational stability. The temperature at the midpoint of thermal denaturation (T(m)) was increased upon modification. Both enthalpy change (DeltaH(m)) and entropy change (DeltaS(m)) for unfolding of modified enzyme at T(m) were decreased compared with native enzyme. Circular dichroism studies proved that these modifications changed the conformation of HRP. The improvements of stability are related to side chain reorientations of aromatics upon both modifications.     

The binding mode of an E-64 analog to the active site of cathepsin B

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhibitor tocathepsin B have been proposed. Molecular docking using an empiricalforce field was carried out to distinguish between the two modes.The search began with manual docking, followed by random perturbationsof the docking conformation and cycles of Monte Carlo minimization.Finally, molecular dynamics was carried out for the most favorabledocking conformations. The present calculations predict thatthe isobutyl-NH-Eps-Leu-Pro inhibitor preferentially binds tothe S' rather than the S subsites of cathepsin B. The S' bindingmode prediction is supported by the X-ray crystal structureof cathepsin B bound to a closely related ethyl-O-Eps-Ile-Proinhibitor, which was found to bind in the S' subsite with theC-terminal epoxy ring carbon making a covalent bond to the sulfuratom of Cys29. This agreement, in turn, validates our dockingstrategy. Furthermore, the calculations provide evidence thatthe dominant contribution to the total stabilization energyof the enzyme–inhibitor complex stems from the strongelectrostatic interaction between the negatively charged C-terminalcarboxylate group of the ligand and the positively charged imidazoliumrings of His110 and His111. The latter are stabilized and heldin an optimal orientation for interactions with the C-terminalend of the ligand through a salt bridge between the side chainsof His110 and Asp22. By comparison with the crystal structure,some insight into the specificity of the epoxyldipeptide familytowards cathepsin B inhibition has been extracted. Both thecharacteristics of the enzyme (e.g. subsite size and hydrophobicity)as well as the nature of the inhibitor influence the selectivityof an inhibitor towards an enzyme.     

Lysozyme requires fluctuation of the active site for the manifestation of activity

Mutations around His15 which lie far away from the active site,stimulated glycol chitin activity of lysozyme at physiologicaltemperature. Del-Argl4Hisl5 lysozyme, a mutant lysozyme whoseArgl4 and Hisl5 were deleted together, and has the highest activityamong these mutant lysozymes, had a similar binding abilityto a trimer of N-acetyl-glucosamine, a substrate analogue, relativeto native lysozyme. This suggests that the increased activitywas due to an increased k_cat in the catalysis reaction. TheH-D exchange rate of the N-1 proton in the Trp63 which is locatedin the active site cleft, was enhanced in the Del-Argl4Hisl5lysozyme, while 2-D proton NMR analysis revealed no conformationalchange around Trp63. We conclude that some sort of fluctuationat the active site might be required for the manifestation ofactivity. This theory is supported by the finding that the Del-Argl4Hisl5lysozyme showed a shift in temperature dependency of activityto lower temperatures compared with that of native lysozyme.     

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.     

Modulation of the enzymatic activity of papain by interdomain residues remote from the active site

The two main catalytic residues Cys25 and Hisl59 of the monomericcysteine protease papain are located on different walls of acleft formed by two domains. This topology suggests a possiblerelationship between relative domain organization and catalyticmechanism. The effect on enzymatic parameters of structuralmodifications at various locations of the twodomain interfaceof papain was examined by individual or double replacementsby Ala of pairs of interacting residues. Most modificationshad no effect on enzyme activity. However, the enzyme's substrateturnover (k_cat) decreased following simultaneous alterationof the two most conserved residues, forming an apolar contactlocated 15 Å away from the active site. The pH activityprofile of the double mutant was unchanged, indicating a conservedionization state of the active site thiolate-imidazolium ionpair. This state is strongly dependent on the distance separatingthe two residues, thus suggesting that the active site geometryhas not been significantly altered. Efficient enzymatic activityin papain requires more than a correct active site geometryand is influenced by domain packing properties in a region remotefrom the active site.