Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease

Oligonucleotide-directed mutagenesis has been used to replaceglycine residues by alanine in neutral protease from Bacillussubtilis. One Gly to Ala substitution (G147A) was located ina helical region of the protein, while the other (G189A) wasin a loop. The effects of mutational substitutions on the functional,conformational and stability properties of the enzyme have beeninvestigated using enzymatic assays and spectroscopic measurements.Single substitutions of both G1y147 and Gly189 with Ala residuesaffect the enzyme kinetic properties using synthetic peptidesas substrates. When Gly replacements were concurrently introducedat both positions, the kinetic characteristics of the doublemutant were roughly intermediate between those of the two singlemutants, and similar to those of the wild-type protease. Bothmutants G147A and G189A were found to be more stable towardsirreversible thermal inactivation/unfolding than the wild-typespecies. Moreover, the stabilizing effect of the Gly to Alasubstitution was roughly additive in the double mutant G147A/G189A,which shows a 3.2°C increase in T_m with respect to the wild-typeprotein. These findings indicate that the Gly to Ala substitutioncan be used as a strategy to stabilize globular proteins. Thepossible mechanisms of protein stabilization are also discussed.     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

Introduction of disulfide bonds into Bacillus subtilis neutral protease

The effects of engineered disulfide bonds on autodigestion andthermostability of Bacillus subtilis neutral protease (NP-sub)were studied using site-directed mutagenesis. After modellingstudies two locations that might be capable of forming disulfidebonds, both near previously determined autodigestion sites inNP-sub, were selected for the introduction of cysteines. Analysisof mutant enzymes showed that disulfide bonds were indeed formedin vivo, and that the mutant enzymes were fully active. Theintroduced disulfides did not alter the autodigestion patternof the NP-sub. All mutant NP-subs exhibited decreased thermostability,which, by using reducing agents, was shown to be caused by theintroduction of the cysteines and not by the formation of thedisulfides. Mutants containing one cysteine exhibited intermoleculardisulfide formation at elevated temperatures, which, however,was shown not to be the cause of the decreased thermostability.Combining the present data with literature data, it would seemthat the introduction of disulfide bridges is unsuitable forthe stabilization of proteases. Possible explanations for thisphenomenon are discussed.     

Stabilization of the neutral protease of Bacillus stearothermophilus by removal of a buried water molecule

Using site-directed mutagenesis, Ala166 in the neutral proteaseof Bacillus stearothermophilus was changed into Ser. Model buildingand molecular dynamics simulations of the mutant enzyme indicatedthat the Ser hydroxyl group fits well in a cavity which containsa water molecule in the wild-type enzyme. The Alal66 - Ser mutationwas expected to exert a stabilizing effect because of the gainin entropy resulting from the release of a water molecule fromthe folded protein to the solvent. In addition, the hydrogen-bondingnetwork around residue 166 was improved upon the mutation. Asa result of this mutation the thermostability of the neutralprotease was increased by 1.2 ± 0.1°C.     

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease

Using genetic techniques the contribution of surface loops tothe thermal stability of Bacillus subtUis neutral protease (NPsub)wasstudied. Mutations were designed to make the surface of NP-submore similar to the surface of more thermostable neutral proteasessuch as thermolysin (TLN). The mutations included the replacementof an irregular loop by a shorter variant and the introductionof a ten–residue (3– hairpin. In general, thesedrastic mutations had little effect on the production and activityof NP–sub, indicating the feasibility of major structuralrearrangements at the surface of proteins. In the most stablemutant, exhibiting an increase in thermal stability of 1.1°C, 10% of the surface of NP–sub was modified. Several NP–subvariants carrying multiple mutations were constructed. Non–additiveeffects on thermal stability were observed, which were interpretedon the basis of a model for thermal inactivation, that emphasizesthe importance of local unfolding processes for thermal stability     

Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Using genetically engineered mutants of the neutral pro-teasefrom Bacillus stearothermophilus (BsteNP), it had been shownthat the surface-exposed structural motif constituted by Phe63embedded in a four amino acid hydrophobic pocket is criticalfor the thermal stability of the thermophilic neutral proteasesfrom Bacilli. To measure the stabilizing contribution of eachhydrophobic interaction taking place between Phe63 and the hydrophobicpocket, we grafted this structural motif in the neutral proteasefrom the mesophile Bacillus subtilis (BsubNP). This was accomplishedby first creating the Thr63Phe mutant of BsubNP and then generatinga series of mutants in which the four amino acids which in thermolysinsurround Phe63 and form the hydrophobic pocket were added oneafter the other. By analysing the thermal stability of eachmutant it was found that the 2°C destabilizing effect ofthe Thr63Phe substitution was completely suppressed by the additionof the four amino acid hydrophobic pocket, each replacementproviding a stabilizing contribution of approxi mately 0.8–1°C.These results are discussed in the light of the peculiar mechanismof thermal inactivation of proteolytic enzymes.     

Mapping of residues involved in the interaction between the Bacillus subtilis xylanase A and proteinaceous wheat xylanase inhibitors

The Bacillus subtilis xylanase A was subjected to site-directed mutagenesis, aimed at changing the interaction with Triticum aestivum xylanase inhibitor, the only wheat endogenous proteinaceous xylanase inhibitor interacting with this xylanase. The published structure of Bacillus circulans XynA was used to target amino acids surrounding the active site cleft of B.subtilis XynA for mutation. Twenty-two residues were mutated, resulting in 62 different variants. The catalytic activity of active mutants ranged from 563 to 5635 XU/mg and the interaction with T.aestivum xylanase inhibitor showed a similar variation. The results indicate that T.aestivum xylanase inhibitor interacts with several amino acid residues surrounding the active site of the enzyme. Three different amino acid substitutions in one particular residue (D11) completely abolished the interaction between T.aestivum xylanase inhibitor and B.subtilis xylanase A.     

A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain

A new high-yield yeast expression/secretion system has beenadapted for the plant thiol endoprotease papain. The propapaingene, obtained from Carica papaya fruit, is expressed in theyeast Saccharomyces cerevisiae. The gene was cloned into a FLAGepitope-tagging expression vector downstream of the yeast alphamating factor (-factor) secretion signal sequence. Expressionof the heterologous propapain in yeast is controlled by theglucose-repressible alcohol dehydrogenase isoenzyme II promoter(ADH2). Glycosylated FLAG-tagged propapain is secreted by asocalled ‘super secretor’ strain, pmr1 (ssc1), intothe culture supernatant where it accumulates to {small tilde}1.7mg/1. The proregion contains three consensus N-linked glycosylationsites, whereas there are only two such sites in previously reportedcDNA sequences. Removal of this third N-linked glycosylationsite results in a drastic reduction in the level of proteaseactivity present in the culture supernatant. Two different typesof affinity chromatography were used to purify either propapainor papain. The propapain precursor is autoproteolytically activatedto mature papain (M_r = 24 kDa) using conditions reported previously.The kinetic parameters obtained agree well with the literaturevalues. The yields of active papain are 10-fold higher thanthose previously reported for propapain in other yeast or bacterialexpression systems. This, together with the ease with whichmutant proteins can be made, makes this yeast advantageous fora structure–function analysis of recombinant wild-typeand mutant papain, and possibly for other related cysteine proteasesas well.     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.     

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Sequential rounds of error-prone PCR to introduce random mutationsand screenrng of the resultant mutant libraries have been usedto enhance the total catalytic activity of subtilisin E significantlyin a non-natural environment, aqueous dimethylformamide (DMF).Seven DNA substitutions coding for three new amino acid substitutionswere identified in a mutant isolated after two additional generationsof directed evolution carried out on 10M subtilisin E, previously‘evolved’ to increase its specific activity in DMF.A Bacillus subtilis-Escherichia coli shuttle vector was developedin order to increase the size of the mutant library that couldbe established in B.subtilis and the stringency of the screeningprocess was increased to reflect total as well as specific activity.This directed evolution approach has been extremely effectivefor improving enzyme activity in a non-natural environment:the resulting-evolved 13M subtilisin exhibits specific catalyticefficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilidein 60% DMF solution that is three times that of the parent 10Mand 471 times that of wild type subtilisin E. The total activityof the 13M culture supernatant is enhanced 16-fold over thatof the parent 10M.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini

Mutations were directed to specific regions of the human tissue-typeplasminogen activator (t-PA) gene in an effort to better definestructure–function relationships of the enzyme. Threetypes of modifications were effected by in vitro mutagenesis:elimination of glycosylation sites; substitutions of amino acidsat the cleavage site for conversion of single-chain t-PA totwo-chain t-PA; and truncations of the N- and C-termini. Thirteenvariants were purified from permanent CHO cell lines and analyzedfor specific activity, fibrin stimulation, fibrin binding, inhibitionby plasminogen activator inhibitor-2 (PAI-2) and half-life.The results of these analyses are: (i) variants with carbohydrate–depletedkringle domains possessed higher specific activities than wild-typet-PA; (ii) a cleavage site variant substituted at Arg275 withGly had greatly reduced specific activity; (iii) two variantssubstituted at Lys277 exhibited altered interactions with PAI-2;(iv) the variant with a truncated C-terminus had reduced activityin the absence of fibrin; and (v) no variants had significantlyaltered half–lives. In order to test the effects of combiningmutations, four additional variants were produced. Each combinationvariant retained at least one of the altered properties observedin the original variants, and in three of the variants the diverseproperties were additive.     

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

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

Identification by site-directed mutagenesis of amino acids in the subsite of bovine pancreatic ribonuclease A

In addition to hydrolysing RNA, bovine pancreatic ribonucleasesplits esters of pyrimidine nucleoside 3'-phosphates, includingdinucleotides. For a series of 3':5'-linked dinucleotides ofgeneral structure CpN, where N is a 5' linked nucleoside, k_calfor the release of N varies enormously with the precise structureof N. Structural studies have been interpreted to indicate thatthe group N interacts with a subsite, B2, on the enzyme thatcomprises Gln69, Asn71 and Glulll. We report studies by site-directedmutagenesis that indicate that Gln69 is not involved in productiveinteractions with any of the dinucleotide substrates and thatAsn71 is an important component of subsite B2 for all dinucleotidesubstrates tested. Glulll appears to be functionally involvedin catalysis for dinucleotide substrates solely when N is guanosine.     

Bipartite organization of the Bacillus subtilis endo-{beta}-1,4-glucanase revealed by C-terminal mutations

The C-terminal boundary of primary sequence of the Bacillussubtilis PAP115 endo-ß-1,4-glucanase (EG) requiredfor stable catalytic activity has been mapped by site-directedmutagenesis using Escherichia coli as host. The 52 kDa cel geneproduct, EG₄₇₀ and a 33 kDa mutant (EG₃₀₀), lacking 170 residuesthrough a nonsense mutation at the leucine-330 codon of thegene, exhibited similar patterns of enzymatic activity and pHoptima using cellooligopentaose as substrate.CD spectra indicatedthat the bulk of the -helical secondary structure in EG₄₇₀ wascontained within EG₃₀₀. However, relative to EG₄₇₀, the specificactivity of EG₃₀₀ was 3- to 4-fold lower with amorphous celluloseas substrate and {small tilde}4-to5-fold higher with carboxymethylcellulose(soluble cellulose).These results along with data which showthat EG₄₇₀ binding capacity to mirocrystalline cellulose is{small tilde} 11 times more than that of EG₃₀₀, demonstratethe importance of residues 330–499 for non-catalytic bindingof cellulose. A construct of the cel gene carrying a deletionof codons 330–499 and an insertion of a nonsense codonat leucine-330, was further used to make mutants EG₂₉₆ and EG₂₉₁with nonsense codon substitutions at arginine and serine-321,respectively.Western analysis using EG-specific antiserum revealedthat relative losses in enzymatic activity of EG₂₉₆ (50%) andEG₂₉₁ (95%) could be accounted for by the extent of their proteolysis,signifying a marked destabilization of these enzymes by removalof only a few amino acids.     

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.     

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.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.