A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 – Cys24, Asn84 – Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min.     

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis

The unusual chemical properties of active-site Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodo-spirillum rubrumhave suggested that this residue is required for catalysis.To test this postulate Lys-329 was replaced with glycine, serine,alanine, cysteine, arginine, glutamic acid or glutamine by site-directedmutagenesis. These single amino acid substitutions do not appearto induce major conformational changes because (i) intersubunitinteractions are unperturbed in that the purified mutant proteinsare stable dimers like the wild-type enzyme and (ii) intrasubunitfolding is normal in that the mutant proteins bind the competitiveinhibitor 6-phosphogluconate with an affinity similar to thatof wild-type enzyme. In contrast, all of the mutant proteinsare severely deficient in carboxylase activity (< 0.01% ofwild-type) and are unable to form the exchange-inert complex,characteristic of the wild-type enzyme, with the transitionstateanalogue carboxyarabinitol bisphosphate. These results underscorethe stringency of the requirement for a lysyl side-chain atposition 329 and imply that Lys-329 is involved in catalysis,perhaps stabilizing a transition state in the overall reactionpathway.     

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

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

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase

A mutant form of pyruvate kinase in which serine 384 has beenmutated to proline has been engineered in the yeast Saccharomycescerevisiae. Residue 384 is located in a helix in a subunit interfaceof the tetrameric enzyme, and the mutation was anticipated toalter the conformation of the helix and hence destabilize theinterface. Previous results indicate that the mutant favoursthe T quarternary conformation over the R conformation, andthis is confirmed by the results presented here. Addition ofphosphoenol-pyruvate (PEP), ADP and fructose-1,6-bisphosphate(Fru 1,6-P₂) singly to the wild-type and mutant enzymes resultsin a significant quenching of tryptophan fluorescence (12–44%),and for Fru-1,6-P₂, a red shift of 15 nm in the emission maximum.Fluorescence titration experiments showed that PEP, ADP andFru-1,6-P₂ induce conformations which have similar ligand-bindingproperties in the wild-type and mutant enzymes. However, theFru-1,6-P₂ induced conformation is demonstrably different fromthose induced by either ADP or PEP. The enzymes differ in theirsusceptibility to trypsin digestion and N-ethylmaleimide inhibition.The thermal stability of the enzyme is unaltered by the mutantion.Far-UV CD spectra show that both enzymes adopt a similar overallsencondary structure in solution. Taken together, the resultssuggest that the Ser384-Pro mutaion causes the enzyme to adopta diffenrent tertiary and/or quaternary structure from the wild-typeenzyme and affects the type and extent of the conformationalchanges induced in the enzyme upon ligand binding. A simplifiedminimal reaction mechanism is proposed in which the R and Tstates differ in both affinity and K_cat. Thus, in terms of themodels of cooperativity and allsoteric interaction, pyruvatekinase is both a K and a V system.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase

Platelet-activating factor acetylhydrolases (PAF-AHs) are uniquePLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipidswith a marked preference for very short acyl chains, typicallyacetyl. The recent solution of the crystal structure of the₁ catalytic subunit of isoform Ib of bovine brain intracellularPAF-AH at 1.7 Å resolution paved the way for a detailedexamination of the molecular basis of substrate specificityin this enzyme. The crystal structure suggests that the sidechains of Thr103, Leu48 and Leu194 are involved in substraterecognition. Three single site mutants (L48A, T103S and L194A)were overexpressed and their structures were solved to 2.3 Åresolution or better by X-ray diffraction methods. Enzyme kineticsshowed that, compared with wild-type protein, all three mutantshave higher relative activity against phospholipids with sn-2acyl chains longer than an acetyl. However, for each of themutants we observed an unexpected and substantial reductionin the V_max of the reaction. These results are consistent withthe model in which residues Leu48, Thr103 and Leu194 indeedcontribute to substrate specificity and in addition suggestthat the integrity of the specificity pocket is critical forthe expression of full catalytic function, thus conferring veryhigh substrate selectivity on the enzyme.     

Activation of E350A mutant maltodextrin phosphorylase by exogenously added acetate

Site-directed mutagenesis of E350 to alanine in Escherichiacoli maltodextrin phosphorylase reduced both enzyme activity(100-fold) and apparent binding of the oligosaccharide substrate(10-fold), suggesting a participation of this residue in bindingof the substrate in the ground and transition states. The E350Amutant enzyme was found to be activated up to 20-fold by exogenousacetate ions which substitute for the deleted side chain. Incontrast, apparent binding was not affected by acetate ions,indicating a dual role for the carboxylic group of this residuein catalysis and binding. Formate also appears to activate theE350A mutant enzyme, but this effect is obscured by the stronginhibitory effect of formate on the wild-type enzyme. For propionateions, a weak 2-fold activation was noticed, while other compoundslike trifluoroacetate and acetamide had no effects on the catalyticproperties of either the E350A mutant enzyme or wild-type enzyme.If E350 was substituted by a glutamine, no activation was observedupon the addition of acetate ions. However, a weak activationby formate was found, confirming that activation by acetateis caused by specific binding at the mutated site.     

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.     

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodexynucleotides

Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the S_Pdiastereomer of the oligodeoxynucleotide GACGATAT_mpCGTC andthe unmodified sequence with equal rates, whereas the R_P diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the R_p diastereomer, but cleaves the S_P diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.     

Chemical modification and site-directed mutagenesis of Tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8

3-Isopropylmalate dehydrogenase from an extreme thermo-phile,Thermus thermophUus HB8, was chemically modified with tetranitromethanewhich nitrated 1.5-2.0 Tyr residues per subunit. The nitrationwas biphask and parallel to the loss of activity. The modifiedresidue in the first phase was identified to be Tyr36, whichis distantly located from the active site of the enzyme. Thefunction of Tyr36 was investigated by site-specific replacementwith Phe. The Michaelis constant for the substrate or co-enzymewas not altered by the replacement, whereas the catalytic constantdecreased down to -5%. X-ray analysis of the mutant enzyme revealedthat Arg94 moved the largest distance among the active siteresidues, that is, the NH1 and NH2 of the guanidino group moved1.11 and 1.32 Å respectively. The results suggest thatArg94 is responsible for the enzyme catalysis     

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.     

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.     

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.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease

On the basis of the homology with the Bacillus thermoproteolyticuszinc endopeptidase thermotysin, we hypothesized that Glu-143and His-231 are the key residues for the catalytic activityof the Bacillus subtilis neutral protease. To test this possibilityby site-directed mutagenesis, we substituted these two residueswith Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively.All these substitutions dramatically affected the amount ofsecreted mutant proteins, as determined by immunological methods,and their catalytic activities. No appreciable secretion wasobserved with the three Glu mutants Trp, Ser and Arg, whereasthe Glu–Ala mutant enzyme was secreted at a level of afew hundred micrograms per litre of culture. The His mutantswere all secreted at higher levels (in the order of a few milligramsper litre) and their residual catalytic activity could be determinedusing Z-Ala-Leu-Ala as substrate. Our results confirm the keyrole played by Glu-143 and His-231 in catalysis and moreoversuggest the existence of a relationship between the catalyticactivity of the enzyme and the extent of its secretion. In thiscontext, we present data suggesting an autoproteolytic mechanismof cleavage of the precursor form of the enzyme, analogous tothe one previously reported for the B.subtilis subtilisin.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidasefrom Streptomyces and that from Brevibacterium, both enzymesexhibit different characteristics, such as catalytic activity,optimum pH and temperature. In attempts to define the molecularbasis of differences in catalytic activity or stability, substitutionsat six amino acid residues were introduced into cholesteroloxidase using site-directed mutagenesis of its gene. The aminoacid substitutions chosen were based on structural comparisonsof cholesterol oxidases from Streptomyces and Brevibacterium.Seven mutant enzymes were constructed with the following aminoacid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357Nand S379T. All the mutant enzymes exhibited activity with theexception of that with the L117P mutation. The resulting V145Qmutant enzyme has low activities for all substrates examinedand the S379T mutant enzyme showed markedly altered substratespecificity compared with the wild-type enzyme. To evaluatethe role of V145 and S379 residues in the reaction, mutantswith two additional substitutions in V145 and four in S379 wereconstructed. The mutant enzymes created by the replacement ofV145 by Asp and Glu had much lower catalytic efficiency forcholesterol and pregnenolone as substrates than the wild-typeenzyme. From previous studies and this study, the V145 residueseems to be important for the stability and substrate bindingof the cholesterol oxidase. In contrast, the catalytic efficiencies(k_cat/K_m) of the S379T mutant enzyme for cholesterol and pregnenolonewere 1.8- and 6.0-fold higher, respectively, than those of thewild-type enzyme. The enhanced catalytic efficiency of the S379Tmutant enzyme for pregnenolone was due to a slightly high k_catvalue and a low K_m value. These findings will provide severalideas for the design of more powerful enzymes that can be appliedto clinical determination of serum cholesterol levels and assterol probes.     

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.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Site-directed mutagenesis was used to alter active-site residuesof methylamine dehydrogenase (MADH) from Paracoccus denitrificans.Four residues of the ß subunit of MADH which are inclose proximity to the tryptophan tryptophylquinone (TTQ) prostheticgroup were modified. The crystal structure of MADH reveals thateach of these residues participates in hydrogen bonding interactionswith other active-site residues, TTQ or water. Relatively conservativemutations which removed the potentially reactive oxygens onthe side chains of Thr122, Tyr119, Asp76 and Asp32 each resultedin greatly reduced or undetectable levels of MADH production.The reduction of MADH levels was determined by assays of activityand Western blots of crude extracts with antisera specific forthe MADH ß subunit. No activity or cross-reactiveprotein was detected in extracts of cells expressing D76N, T122Aand T122C MADH mutants. Very low levels of active MADH wereproduced by cells expressing D32N, Y119F, Y119E and Y119K MADHmutants. The Y119F and D32N mutants were purified from cellextracts and found to be significantly less stable than wild-typeMADH. Only the T122S MADH mutant was produced at near wild-typelevels. Possible roles for these amino acid residues in stabilizingunusual structural features of the MADH ß subunit,protein folding and TTQ biosynthesis are discussed.     

An engineered Staphylococcus aureus PCI {beta}-lactamase that hydrolyses third-generation cephalosporins

The ß-lactamase from Staphylococcus aureus PCI hasbeen cloned into an Escherichia coli vector for site-directedmutagenesis and high-level protein expression. A mutant enzymehas been produced in which Ala238 is replaced by a serine, andIle239 is deleted (A238S:I239del). The engineered enzyme hydrolysesthird-generation cephalosporins substantially more rapidly thanthe parental enzyme does, while hydrolysis of benzylpenicillinis slower with the mutant than with the wild-type and nativeenzymes. The mutant P-lactamase has been crystallized and thestructure determined and refined at 2.8 A resolution. The dispositionof the ß-strand which forms the side of the activesite is altered in comparison with the native S.aureus ß-lactamasestructure, widening the active site cleft and providing spaceto accommodate the bulky side-chains of the third-generationcephalosporins.     

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.     

Protein engineering of alcohol dehydrogenase -- 1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the abilityto alter predictably the properties of an enzyme to enable itto find new substrates or catalyse existing substrates moreefficiently, such manipulations being of interest both enzymologicallyand, potentially, industrially. It has been postulated thatin yeast alcohol dehydrogenase (YADH-1) certain amino acidssuch as Trp 93 and Thr 48 constrict the active site due to theirbulky side chains and thus impede catalysis of molecules largerthan ethanol. To study effects of enlarging the active sitewe have made two changes into YADH-1, replacing Trp 93 withPhe and Thr 48 with Ser. Kinetic experiments showed that thisenzyme had marked increases in reaction velocity for the n-alcoholspropanol, butanol, pentanol, hexanol, heptanol, octanol andcinnamyl alcohol compared to the parent, agreeing with the predictionthat expanding the active site should facilitate the oxidationof larger alcohols. The substrate affinities were slightly reducedin the altered enzyme, possibly due to its having reduced hydrophobicityat Phe 93.