Theoretical examination of the mechanism of aldose-ketose isomerization

Two mechanisms for an aldose–ketose isomerization havebeen examined using high level ab initio and semiempirical molecularorbital methods. The proton transfer pathway via an enediolintermediate is shown to be favored in the absence of a metalion, while the hydride transfer pathway becomes favored in thepresence of a metal ion. Our calculations explain why the protontransfer pathway is operative in most aldose–ketose isomerizationreactions. These calculations also provide further support forthe previously proposed metal ion-mediated hydride transfermechanism for xylose isomerase.     

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

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

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.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5 [EC] ] fromStreptomyces olivochromogenes has been determined to 3.0 Åresolution. The crystals belong to space group P22₁2₁ with unitcell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetricunit contains half of a tetrameric molecule of 222 symmetry.The two-fold axis relating the two molecules in the asymmetricunit is close to where a crystallographic two-fold would beif the space group were 1222. This causes the diffraction patternto have strong 1222 pseudo-symmetry, so all data were collectedin this pseudo-space group. Since the sequence of this enzymehas not been reported, a polyalanine backbone has been fittedto the electron density. Xylose isomerase has two domains: theN-terminal domain is an eight-stranded /ß barrel of299 residues. The C-terminal domain is a large loop of 50 residueswhich is involved in inter-molecular contacts. Comparison ofxylose isomerase with the archetypical /ß barrel protein,triose phosphate isomerase, reveals that the proteins overlapbest when the third (ß) strand of xylose isomeraseis superimposed on the first (ß) strand of triosephosphate isomerase. This same overlap has also been found betweenthe muconate lactonising enzyme and triose phosphate isomerase[Goldman et al. (1987) J. Mol. Biol., in press].     

Engineering the substrate specificity of xylose isomerase

Xylose isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

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.     

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

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

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.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.     

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

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.     

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.     

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.     

Engineering a heavy atom derivative for the X-ray structure analysis of cyclodextrin glycosyltransferase

Based on a preliminary structural model of cyclodextrin glycosyltransferasefrom Bacillus circulans (EC 2.4.1.19 [EC] ), Ser428 and Ser475 ofthe enzyme were mutated to cysteines in order to produce suitableheavy atom derivatives. Mutant Ser475 - Cys could not be expressedas protein. Mutant Ser428 - Cys was expressed in Escherichiacoli and purified. It crystallized isomorphously and gave riseto a mercury derivative that improved the electron density map.The structural results show that the new mercury-binding siteis in a pocket at the protein surface.     

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide

The 2.9 Å resolution structure of iron superoxide dismutase(FeSOD) (EC 1.15.1.1 [EC] ) from Pseudomonas ovalis complexed withthe inhibitor azide was solved. Comparison of this structurewith free enzyme shows that the inhibitor is bound at the opencoordination position of the iron, with a bond length of 2.0Å. The metal moves by 0.4 Å into the trigonal planeto produce an orthogonal geometry at the iron. Binding of theinhibitor also causes a movement of the axial ligand (histidine26) away from the metal, a lengthening of the iron—histidinebond, and a rotation of the histidine 74 ring. The inhibitorpossesses contacts in the binding pocket with a pair of conservedtryptophan residues and with the side chains of tyrosine 34and glutamine 70. This glutamine is conserved between all FeSODs,but is absent in MnSOD. Comparisons with MnSOD show that a differentglutamine which possesses the same interactions in the activesite as Gln70 in FeSOD is conserved at position 154 in the overallSOD sequence, implying that while manganese and FeSODs are structuralhomologues in a global sense, their functional and evolutionaryrelationship is that of second-site mutation revertants.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic andcarboxamide groups in proteins is presented. Most of the ligandsare from aspartic and glutamic acid side chains. Water moleculesbound to carboxylate anions are known to interact with oxygenlone-pairs. However, metal ions are also found to approach thecarboxylate group along the C - O direction. More metal ionsare found to be along the syn than the anti lone-pair direction.This seems to be the result of the stability of the five-memberedring that is formed by the carboxylate anion hydrogen bondedto a ligand water molecule and the metal ion in the syn position.Ligand residues are usually from the helix, turn or regionswith no regular secondary structure. Because of the steric interactionsassociated with bringing all the ligands around a metal center,a calcium ion can bind only near the ends of a helix; a metal,like zinc, with a low coordination number, can bind anywherein the helix. Based on the analysis of the positions of watermolecules in the metal coordination sphere, the sequence ofthe EF hand (a calcium-binding structure) is discussed.     

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase

The large form of glucoamylase (GAI) from Aspergillus awamori(EC 3.2.1.3 [EC] ) binds strongly to native granular starch, whereasa truncated form (GAII) which lacks 103 C-terminal residues,does not. This C-terminal region, conserved among fungal glucoamylasesand other starch-degrading enzymes, is part of an independentstarch-binding domain (SBD). To investigate the SBD boundariesand the function of conserved residues in two putative substrate-bindingsites, five gluco-amylase mutants were constructed with extensivedeletions in this region for expression in Saccharomyces cerevisiae.Progressive loss of both starch-binding and starch-hydrolyticactivity occurred upon removal of eight and 25 C-terminal aminoacid residues, or 21 and 52 residues close to the N-terminus,confirming the requirement for the entire region in formationof a functional SBD. C-terminal deletions strongly impairedSBD function, suggesting a more important role for one of theputative binding sites. A GAII phenocopy showed a nearly completeloss of starch-binding and starch-hydrolytic activity. The deletionsdid not affect enzyme activity on soluble starch or thermo-stabilityof the enzyme, confirming the independence of the catalyticdomain from the SBD.