Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86)

The moderate thermophile Bacillus stearothermophilus was usedas a host in which to detect more thermostable variants of theB.pumilus chloramphenicol acetyltransferase (Cat-86) protein.Seventeen mutants were isolated and detected by their abilityto grow in the presence of chloramphenicol at a previously restrictivetemperature (58°C). The genes encoding these proteins weresequenced; all 17 mutants carried the same C to T transitionthat conferred an amino acid substitution of alanine by valineat position 203 of the protein sequence. The wild-type and onemutant Cat-86 protein were purified to homogeneity using affinitychromatography, and kinetic and thermal stability studies wereundertaken. Both enzymes had similar sp. act. in the regionof 215 U/mg, with K_m values for chloramphenicol in the range13.8–15.4 µM and for acetyl CoA in the range 13.6–15.5µM. The A203V mutant shows greater stability than thewild-type Cat-86 protein at temperatures above 50°C andappears to pass through a transition state between 48 and 50°C.     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisinE into an enzyme functionally equivalent to its thermophilichomolog thermitase from Thermoactinomyces vulgaris. Five generationsof random mutagenesis, recombination and screening created subtilisinE 5-3H5, whose half-life at 83°C (3.5 min) and temperatureoptimum for activity (T_opt, 76°C) are identical with thoseof thermitase. The T_opt of the evolved enzyme is 17°C higherand its half-life at 65°C is >200 times that of wild-typesubtilisin E. In addition, 5-3H5 is more active towards thehydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-typeat all temperatures from 10 to 90°C. Thermitase differsfrom subtilisin E at 157 amino acid positions. However, onlyeight amino acid substitutions were sufficient to convert subtilisinE into an enzyme equally thermostable. The eight substitutions,which include known stabilizing mutations (N218S, N76D) andalso several not previously reported, are distributed over thesurface of the enzyme. Only two (N218S, N181D) are found inthermitase. Directed evolution provides a powerful tool to unveilmechanisms of thermal adaptation and is an effective and efficientapproach to increasing thermostability without compromisingenzyme activity.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189–338, M_r = 16 222) of glutamatedehydrogenase from the hyperthermophilic bacterium Thermotogamaritima was used as a model system to study reversible unfoldingthermodynamics of this hyperthermostable enzyme. The proteinwas produced in large quantities in E.coli using a T7 expressionsystem. It was shown that the recombinant domain is monomericin solution and that it comprises secondary structural elementssimilar to those observed in the crystal structure of the hexamericenzyme.The recombinant domain is thermostable and undergoesreversible and cooperative thermal unfolding in the pH range5.90–8.00 with melting temperatures between 75.1 and 68.0°C.Thermal unfolding of the protein was studied using differentialscanning calorimetry and circular dichroism spectroscopy. Bothmethods yielded comparable values. The analysis revealed anunfolding enthalpy at 70°C of 70.2 ± 4.0 kcal/moland a     

Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region

The thermostability enhancement of Flavobacterium meningosepticumglycerol kinase (FGK) by random mutagenesis in the subunit interfaceregion was investigated. A single Escherichia coli transformant,which produced a more thermostable glycerol kinase than theparent enzyme, was obtained. The nucleotide sequence of thegene of the mutant enzyme (FGK2615) was determined, and thefour amino acid replacements were identified as Glu327 to Asp,Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although theproperties of FGK2615 were fundamentally similar to those ofthe parent enzyme, the thermostability and K_m for ATP had changed.The thermostability of FGK2615 was apparently increased; thetemperature at which the enzyme activity is inactivated by 50%for a 30-min incubation of FGK2615 was determined to be 72.1°Cwhich was 3.1°C higher than that of the parent FGK. Fouradditional mutants each having a single amino acid replacement(Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys)were prepared and their thermostability and K_m for substrateswere evaluated. The effect of the substitution of Ser329 toAsp is discussed.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from13 homologous fungal phytases. A synthetic gene was constructedand recombinantly expressed. Surprisingly, consensus phytase-1was 15–26°C more thermostable than all parent phytasesused in its design [Lehmann et al. (2000)Protein Eng., 13, 49–57].In the present study, inclusion of six further phytase sequencesin the amino acid sequence alignment resulted in the replacementof 38 amino acid residues in either one or both of the new consensusphytases-10 and -11. Since consensus phytase-10, again, was7.4°C more thermostable than consensus phytase-1, the thermostabilityeffects of most of the 38 amino acid substitutions were testedby site-directed mutagenesis. Both stabilizing and destabilizingmutations were identified, but all affected the stability ofthe enzyme by <3°C. The combination of all stabilizingamino acid exchanges in a multiple mutant of consensus phytase-1increased the unfolding temperature from 78.0 to 88.5°C.Likewise, back-mutation of four destabilizing amino acids andintroduction of an additional stabilizing amino acid in consensusphytase-10 further increased the unfolding temperature from85.4 to 90.4°C. The thermostabilization achieved is theresult of a combination of slight improvements from multipleamino acid exchanges rather than being the effect of a singleor of just a few dominating mutations that have been introducedby chance. The present findings support the general validityof the consensus concept for thermostability engineering ofproteins.     

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis

Within the BRIDGE T-project on lipases we investigate the structure-functionrelationships of the lipases from Bacillus subtilis and Pseudomonasaeruginosa. Construction of an overproducing Bacillus. strainallowed the purification of > 100 mg lipase from 30 l culturesupernatant. After testing a large variety of crystallizationconditions, the Bacillus lipase gave crystals of reasonablequality in PEG-4000 (38-45%), Na₂SO₄ and octyl-ß-glucosideat 22°C, pH 9.0. A 2.5 Å; dataset has been obtainedwhich is complete from 15 to 2.5 A resolution. P.aeruginosawild-type strain PAC1R was fermented using conditions of maximumlipase production. More than 90% of the lipase was cell boundand could be solubilized by treatment of the cells with TritonX-100. This permitted the purification of 50 mg lipase. So far,no crystals of sufficient quality were obtained. Comparisonof the model we built for the Pseudomonas lipase, on the basisof sequences and structures of various hydrolases which werefound to possess a common folding pattern (/ß hydrolasefold), with the X-ray structure of the P.glumae lipase revealedthat it is possible to correctly build the structure of thecore of a protein even in the absence of obvious sequence homologywith a protein of known 3-D structure.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

The rnhA gene encoding RNase HI from a psychrotrophic bacterium,Shewanella sp. SIB1, was cloned, sequenced and overexpressedin an rnh mutant strain of Escherichia coli. SIB1 RNase HI iscomposed of 157 amino acid residues and shows 63% amino acidsequence identity to E.coli RNase HI. Upon induction, the recombinantprotein accumulated in the cells in an insoluble form. Thisprotein was solubilized and purified in the presence of 7 Murea and refolded by removing urea. Determination of the enzymaticactivity using M13 DNA–RNA hybrid as a substrate revealedthat the enzymatic properties of SIB1 RNase HI, such as divalentcation requirement, pH optimum and cleavage mode of a substrate,are similar to those of E.coli RNase HI. However, SIB1 RNaseHI was much less stable than E.coli RNase HI and the temperature(T_1/2) at which the enzyme loses half of its activity upon incubationfor 10 min was ~25°C for SIB1 RNase HI and ~60°C forE.coli RNase HI. The optimum temperature for the SIB1 RNaseHI activity was also shifted downward by 20°C compared withthat of E.coli RNase HI. Nevertheless, SIB1 RNase HI was lessactive than E.coli RNase HI even at low temperatures. The specificactivity determined at 10°C was 0.29 units/mg for SIB1 RNaseHI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesisstudies suggest that the amino acid substitution in the middleof the     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumiluschloramphenicol acetyltransferase (CAT-86) to determine theeffects of substitution at a conserved hydrophobic pocket identifiedearlier as important for thermostability. Mutations were introducedthat would substitute residues at consensus positions 33, 191and 203 in the enzyme, both individually and in combination.Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I),were more thermostable than wild-type and two mutants, SDM4(CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstructionof the residues of this hydrophobic pocket to that of a morethermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203Vtriple mutant) increased the thermostability of the enzyme abovethe wild-type, but its stability was less than that of SDM1and SDM5. The K_m values of the mutant enzymes for chloramphenicoland acetyl-CoA were essentially unaltered (in the ranges 15–30and 26–35 µM respectively) and the specific activityof purified enzyme was in the range 270–710 units/mg protein.The possible effects of the amino acid substitutions on theCAT-86 structure were determined by homology modelling. A reductionin conformational strain and optimized hydrophobic interactionsare predicted to be responsible for the increased thermostabilityof the SDM1 and SDM5 mutants.     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-gal–proAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor X_a. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-gal–proAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor X_a digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorX_a, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDS–polyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologousand highly resolved protein crystal structures were used toidentify side-chain main-chain (SC-MC) hydrogen bonds. The ratioof the number of SC-MC hydrogen bonds to the total number ofpolar residues is close to 1:2, indicating the ubiquitous natureof such hydrogen bonds. Close to 56% of the SC-MC hydrogen bondsare local involving side-chain acceptor/donor (`i') and a main-chaindonor/acceptor within the window i–5 to i+5. These short-rangehydrogen bonds form well defined conformational motifs characterizedby specific combinations of backbone and side-chain torsionangles. (a) The Ser/Thr residues show the greatest preferencein forming intra-helical hydrogen bonds between the atoms O_iand O_i–4. More than half the examples of such hydrogenbonds are found at the middle of -helices rather than at theirends. The most favoured motif of these examples is _RRRR(g^–).(b) These residues also show great preference to form hydrogenbonds between O_i and O_i–3, which are closely related tothe previous type and though intra-helical, these hydrogen bondsare more often found at the C-termini of helices than at themiddle. The motif represented by _RRRR(g⁺) is most preferredin these cases. (c) The Ser, Thr and Glu are the most frequentlyfound residues participating in intra-residue hydrogen bonds(between the side-chain and main-chain of the same residue)which are characterized by specific motifs of the form ß(g⁺)for Ser/Thr residues and _R(g^–g⁺t) for Glu/Gln. (d) Theside-chain acceptor atoms of Asn/Asp and Ser/Thr residues showhigh preference to form hydrogen bonds with acceptors two residuesahead in the chain, which are characterized by the motifs ß (tt')Rand ß(t)_R, respectively. These hydrogen bonded segments,referred to as Asx turns, are known to provide stability totype I and type I' ß-turns. (e) Ser/Thr residues oftenform a combination of SC-MC hydrogen bonds, with the side-chaindonor hydrogen bonded to the carbonyl oxygen of its own peptidebackbone and the side-chain acceptor hydrogen bonded to an amidehydrogen three residues ahead in the sequence. Such motifs arequite often seen at the beginning of -helices, which are characterizedby the ß(g⁺)_RR motif. A remarkable majority of all thesehydrogen bonds are buried from the protein surface, away fromthe surrounding solvent. This strongly indicates the possibilityof side-chains playing the role of the backbone, in the proteininteriors, to satisfy the potential hydrogen bonding sites andmaintaining the network of hydrogen bonds which is crucial tothe structure of the protein.     

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.     

Second-generation octarellins: two new de novo ({beta}/{alpha})8 polypeptides designed for investigating the influence of {beta}-residue packing on the {alpha}/{beta}-barrel structure stability

The sequence of octarellin I, the first de novo (ß/)₈polypeptide, was revised according to several criteria, amongothers the symmetry of the sequence, ß-residue volumeand hydrophobicity, and charge distribution. These considerationsand the overall conclusions drawn from the first design ledto two new sequences, corresponding to octarellins II and III.Octarellin II retains perfect 8-fold symmetry. Octarellin IIIhas the same sequence as octarellin II, except for the ß-strandswhich exhibit a 4-fold symmetry. The two proteins were producedin Escherichia coli. Infrared and CD spectral analyses of octarellinsII and III reveal a high secondary structure content. Non-denaturinggel electrophoresis, molecular sieve chromatography and analyticalultracentrifugation suggest that both of these second-generationartificial polypeptides exist as a mixture of a monomer anda dimer form. Octarellins II and III are at least 10 times moresoluble than octarellin I. Ureainduced unfolding followed byfluorescence emission suggests that the tryptophan residues,designed to be buried in the (ß/)₈, are indeed packedin the hydrophobic core of both proteins. However, octarellinIII displays a higher stability towards urea denaturation, indicatingthat introducing 4-fold symmetry into the ß-barrelmight be important for stability of the overall folding.     

Characterization of glycosylated variants of {beta}-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of ß-lactoglobulin (BLG) wereproduced in the methylotrophic yeast Pichia pastoris to mimicthe glycosylation pattern of glycodelin, a homologue of BLGfound in humans. Glycodelin has three sites for glycosylation,corresponding to amino acids 63–65 (S1), 85–87 (S2)and 28–30 (S3) of BLG. These three sites were engineeredinto BLG to produce the variants S2, S12 and S123, which carriedone, two and three glycosylation sites, respectively. The oligosaccharideson these BLG variants ranged from (mannose)₉(N-acetylglucosamine)₂(Man₉GN₂) to Man₁₅GN₂ and were of the     

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticusDNA polymerase (Taq polymerase), which has no catalytic activity,has been exchanged for the 3'–5' exonuclease domain ofthe homologous mesophile Escherichia coli DNA polymerase I (E.colipol I) and the homologous thermostable Thermotoga neapolitanaDNA polymerase (Tne polymerase). Three chimeric DNA polymeraseshave been constructed using the three-dimensional (3D) structureof the Klenow fragment of the E.coli pol I and 3D models ofthe intervening and polymerase domains of the Taq polymeraseand the Tne polymerase: chimera TaqEc1 (exchange of residues292–423 from Taq polymerase for residues 327–519of E.coli pol I), chimera TaqTne1 (exchange of residues 292–423of Taq polymerase for residues 295–485 of Tne polymerase)and chimera TaqTne2 (exchange of residues 292–448 of Taqpolymerase for residues 295–510 of Tne polymerase). Thechimera TaqEc1 showed characteristics from both parental polymerasesat an intermediate temperature of 50°C: high polymeraseactivity, processivity, 3'–5' exonuclease activity andproof-reading function. In comparison, the chimeras TaqTne1and TaqTne2 showed no significant 3'–5' exonuclease activityand no proof-reading function. The chimera TaqTne1 showed anoptimum temperature at 60°C, decreased polymerase activitycompared with the Taq polymerase and reduced processivity. Thechimera TaqTne2 showed high polymerase activity at 72°C,processivity and less reduced thermostability compared withthe chimera TaqTne1.     

Improvement of Oxidative and Thermostability of N-Carbamyl-D-Amino Acid Amidohydrolase by Directed Evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), whichis currently employed in the industrial production of unnaturalD-amino acid in conjunction with D-hydantoinase, has low oxidativeand thermostability. We attempted the simultaneous improvementof the oxidative and thermostability of N-carbamoylase fromAgrobacterium tumefaciens NRRL B11291 by directed evolutionusing DNA shuffling. In a second generation of evolution, thebest mutant 2S3 with improved oxidative and thermostabilitywas selected, purified and characterized. The temperature atwhich 50% of the initial activity remains after incubation for30 min was 73°C for 2S3, whereas it was 61°C for wild-typeenzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxidefor 30 min at 25°C resulted in a complete loss of activity,but 2S3 retained about 79% of the initial activity under thesame conditions. The K_m value of 2S3 was estimated to be similarto that of wild-type enzyme; however k_cat was decreased, leadingto a slightly reduced value of k_cat/K_m, compared with wild-typeenzyme. DNA sequence analysis revealed that six amino acid residueswere changed in 2S3 and substitutions included Q23L, V40A, H58Y,G75S, M184L and T262A. The stabilizing effects of each aminoacid residue were investigated by incorporating mutations individuallyinto wild-type enzyme. Q23L, H58Y, M184L and T262A were foundto enhance both oxidative and thermostability of the enzymeand of them, T262A showed the most significant effect. V40Aand G75S gave rise to an increase only in oxidative stability.The positions of the mutated amino acid residues were identifiedin the structure of N-carbamoylase from Agrobacterium sp. KNK712 and structural analysis of the stabilizing effects of eachamino acid substitution was also carried out.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method

A temperature-jump (T-jump) time-resolved X-ray crystallographictechnique using the Laue method was developed to detect small,localized structural changes of proteins in crystals exposedto a temperature increase induced by laser irradiation. In achimeric protein between thermophilic and mesophilic 3-isopropylmalatedehydrogenases (2T2M6T), the initial structural change uponT-jump to a denaturing temperature (~90°C) was found tobe localized at a region which includes a ß-turn and aloop located between the two domains of the enzyme. A mutant,2T2M6T-E110P/S111G/S113E, having amino acid replacements inthis ß-turn region with the corresponding residues ofthe thermophilic enzyme, showed greater stability than the originalchimera (increase of T_m by ~10°C) and no T-jump-inducedstructural change in this region was detected by our method.These results indicate that thermal unfolding of the originalchimeric enzyme, 2T2M6T, is triggered in this ß-turn region.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

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.     

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