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.     

Molecular dynamics simulations of active site mutants of triosephosphate isomerase

Molecular dynamics simulations of triosephosphate isomerase(TIM) and of some active site TIM mutants were performed inan attempt to elucidate possible interactions important forcatalytic activity and binding. A variety of active site residuesin TIM have been altered, resulting in all cases in decreasesin catalytic activity. Second-site suppressor mutants were characterizedfor two of these active site mutants. The pseudorevertants haveincreased activity compared to the single mutant from whichthey were derived and, surprisingly, in both cases the increasehi activity is a result of the replacement of an active siteserine for proline. We performed simulations of wild-type TIMand the active site mutants with the substrate dihydroxyacetonephosphate bound both non-covalently and covalently. The noncovalentcomplexes were used to examine interactions important to bindingwhile the covalent complexes are models of the transition statestructure for enolization, which is the rate-determining stepfor the mutants. The difference between these two states, then,is related to the catalytic activity. We found various protein-substrateinteractions that unproved in the noncovalent mutant complexes,which correlates with the experimentally observed increase inbinding affinity upon mutation. In the covalent complexes weobserved improved electrostatic stabilization of the transitionstate upon introduction of Pro, which is also consistent withthe experimental data. Our simulations reproduce the highlyco-operative nature of the interactions in the active site andsuggest that this approach may be useful for identifying particularlypromising sites for mutation.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin.     

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

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

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability

To eludicate the role in conformational stability of Cys residuesburied in the interior of a protein, the thermodynamic propertiesof denaturation of mutant subunit of Escherichia coli tryptophansynthase, in which Ser, Ala, Val or Gly was substituted foreach of the three Cys residues, were analyzed using calorimetry.The mutants were less stable than the wild type, indicatingthat Cys residues contribute greatly to the stability of the subunit. In most cases, a large decrease in denaturation enthalpywas observed, compensated for by the denaturation entropy toa major extent. The extent of changes in the denaturation Gibbsenergy and denaturation enthalpy varied greatly depending onboth substituting residues and positions. Of all the mutantproteins, the Cys154Ser mutant showed the greatest decreasein denaturation enthalpy; its denaturation enthalpy was halfthat of the wild type, and was considerably repaired by addinga ligand of the subunit. Because the enthalpy of ligand bindingto Cys154Ser in the native state did not change. it seems thatthe decrease in the denaturation enthalpy of Cys154Ser and itsrecovery by ligand binding are caused by conformational changesin the denatured state due to the mutation.     

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

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

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat

The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of –170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the k_cat/k_m ratio were observed.     

Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (k_cat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (K_M) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 – Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 – 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 – Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2.     

Comparison of backbone structures of glucose isomerase from Streptomyces and Arthrobacter

The C backbones of the glucose isomerase molecules of Streptomycesrubiginosus and Arthrobacter have been determined by X-ray crystallographyand compared. Each molecule is a tetramer of eight-stranded/ß barrels, and the mode of association of the tetramersis identical in each case. The Arthrobacter electron densityshows four additional amino acids at the carboxyl terminus.There is also an insertion of six amino acids at position 277,and two individual insertions at about positions 348 and 357(numbering according to the Streptomyces structure). There isa close structural homology throughout the whole molecule, whichis most accurate up to position 325. The r.m.s. displacementfor 315 homologous C positions up to this position is 0.92 Å.     

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.     

Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis

Crystal structures are known for aspartate carbamoyltransferase(ATCase) in the T and R states, with and without the allostericactivator adenosine triphosphate (ATP) or inhibitor cytidinetriphosphate (CTP). Visual inspection of X-ray crystal structuresdoes not provide all of the information necessary for the determinationof structure-function relationships in protein molecules. Thisproblem is compounded because the crystalline states of themolecule may introduce effects due to crystal packing, restrictedflexibility and less than optimum enzymatic conditions. Therefore,alternative techniques are required to test mechanisms conjecturedfrom three-dimensional crystal structures of proteins. The techniqueof site-specific mutagenesis allows the researcher to test structure- function models based on threedimensional structures and toobtain further insight into characteristics of the enzyme. Site-specificmutagenesis has been used to probe residues believed to be criticalin the structure and function of ATCase. Selection of residuesto be mutated has depended extensively on three-dimensionalcrystal structures of the enzyme. To date, 48 site-specificmutations at 37 different amino acid sites have been published.Although a total of 118 mutants at 58 different sites has beencommunicated to our laboratory, only published mutants willbe considered in this review. In this paper, we compile forthe first time, review, and analyze the site-specific mutantsof ATCase. Site-specific mutagenesis of proteins has becomea powerful technique in modern-day molecular biology, especiallyin studying a molecule as large as aspartate carbamoyltransferase.In this review, the role of site-specific mutagenesis of ATCaseis discussed and improvements in the analysis are suggested.     

Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability

Systematic replacement of the amino acid residues in Escherichiacoli ribonuclease HI with those in the thermophilic counterparthas revealed that two mutations, His62–Pro (H62P) andLys95Gly (K95G), increased the thermostability of the protein.These single-site mutant proteins, together with the mutantproteins His62Ala (H62A), Lys95Asn (K95N) and Lys95Ala (K95A),were crystallized and their structures were determined at 1.8Å resolution. The crystal structures of these mutant proteinsreveal that only the local structure around each mutation siteis essential for the increase in thermostability. For each mutantprotein, the stabilization mechanism is considered to be asfollows: (i) H62P is stabilized because of a decrease in theentropy of the unfolded state, without a change in the nativebackbone structure; (ii) K95G is stabilized since the straincaused by the left-handed backbone structure in the typical3:5 type loop is eliminated; and (iii) K95N is slightly stabilizedby a hydrogen bond formed between the side-chain N-atom of themutated aspargine residue and the main-chain carbonyl oxygenwithin the same residue.     

Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins

The phosphocarrier HPr (heat stable protein) of Staphylococcuscarnosus was modified by site-directed mutagenesis of the correspondingptsH gene in order to analyse the importance of amino acidswhich were supposed to be part of the active centre of the protein.Three residues which are conserved in all HPrs, Argl7, Prol8and Glu84, were mutated: Argl7 was changed to His (17RH) andPro18 and Glu84 were changed into Ala (18PA and 84EA). In addition,Leu86 was changed into Ala (86LA) and one mutant protein wasmissing the last six residues of the HPr (83). The wild typegene and all mutant genes were overexpressed and the gene productspurified to homogeneity. Three-dimensional structures of wildtype and mutant proteins were monitored by NMR spectroscopy.All five mutant HPrs had native conformations. The ATP-dependentHPr kinase can phosphorylate all HPr derivatives at Ser46. ThePTS activity of the amino-terminal HPr mutant proteins 17RHand 18PA was different compared to wild type HPr. In contrast,the car boxy-terminal mutant HPrs possessed a similar enzymeactivity to the wild type HPr. The 17RH and 18PA HPrs with substitutionnear the active centre His15 showed a very slow phosphorylationby enzyme I but the further transfer of the phosphoryl groupto enzyme III was also strongly inhibited. The enzyme activityof the HPr 17RH was significantly improved at low pH. NMR pH-titrationexperiments showed that Arg17 is not responsible for the lowpK_a, of the active centre His15 but this positively chargedresidue is essential in this position for the HPr activity.     

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

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

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.     

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.     

Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases

The pregnancy-associated glycoproteins (PAGs) are secretoryproducts synthesized by the outer epithelial cell layer (chorion)of the placentas of various ungulate species. The amino acidsequences of eight PAGs have been inferred from cloned cDNAof cattle and sheep, as well as of the non-ruminant pig andhorse. We compare the PAG sequences and present results of thethree-dimensional models of boPAG-1 and ovPAG-1 that were constructedon the basis of the crystal structures of homologous porcinepepsin and bovine chymosin using a rule-based comparative modellingapproach. Further, we compare peptide binding subsites definedby interactions with pepstatin and a decapeptide inhibitor (CH-66)modelled on the basis of crystal structures of other asparticproteinases. We have extended our analysis of the peptide bindingsubsites to the other PAG molecules of known sequence by aligningthe PAG sequences to the structural template derived from thepepsin family and by making use of the three-dimensional modelsof the boPAG-1 and ovPAG-1. The residues that are likely toaffect peptide binding in the boPAG-1, ovPAG-1 and other PAGmolecules have been identified. Sequence comparisons revealthat all PAG molecules may have evolved from a pepsin-like progenitormolecule with the equine PAG most closely related to the pepsins.The presence of substitutions at the S₁ and other subsites relativeto pepsin make it unlikely that either bovine, ovine or theporcine PAG-1 have catalytic activity. Only two of the eightPAGs examined (porcine PAG-2 and equine PAG-1) retain featuresof active aspartic proteinases with pepsin-like activity. Ourresults indicate that in the PAGs so far characterized the peptidebinding specificities differ significantly from each other andfrom pepsin, despite their high sequence identities. Analysisof the various peptide binding subsites demonstrates why bothbovine and ovine PAG-1 are capable of binding pepstatin. Thestrong negative charge in the binding cleft of boPAG-1 and ovPAG-1indicates a preference for lysine- or arginine-rich peptides.PAGs represent a family where the possible peptide binding functionmay be retained through their binding specificities, but wherethe catalytic activity may be lost in some cases, such as theboPAG-1, ovPAG-1 and the poPAG-1.     

Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1and -2 (TIMP-1 and -2), comprising the N-terminal active domain,are ideal molecules for structural analysis by intrinsic fluorescenceas each contains a single conserved tryptophan residue. In thispaper we describe studies on their conformational stability,unfolding/refolding kinetics and the environment of the uniquetryptophan as judged by its fluorescence properties in the nativestate and exposure to an external quencher, acrylamide. Twoforms of TIMP-2 were studied: TIMP-2 T21 derived from the full-lengthcDNA clone isolated from a mixed-tumour library, and TIMP-2A21 containing the highly conserved V18IRAK22 sequence. In allthree TIMP proteins the tryptophan environments in the nativestate appeared to be similar, but substantial differences wereseen in their conformational stabilities and refolding kinetics.TIMP-1 was approximately twice as stable as TIMP-2 T21 and 1.4-foldmore stable than TIMP-2 A21. This stability difference betweenTIMP-1 and TIMP-2 was shown to be independent of N-linked glycosylation.TTMP-1 and TIMP-2 A21 both showed simple two-state refoldingkinetics, whereas TIMP-2 T21 refolding was more complex andbiphasic in character. These differences between TIMP-2 T21and A21 suggest that residue 21 is a structurally importantsite in the TIMP protein.All three truncated molecules can beconsidered as stable independent folding domains ideally suitedfor further structural analysis     

Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy

Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.     

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6

Pseudoazurin, a low molecular weight protein containing a singletype I copper, functions as an electron donor to a copper-containingnitrite reductase (NIR) in a denitrifying bacterium Alcaligenesfaecalis S-6. To elucidate the proteinprotein interaction betweenthese two copper-containing proteins, each of nine out of 13lysine residues on the surface of pseudoazurin were independentlyreplaced by alanine or aspartate, and the effects of the mutationson the interaction with NIR, as well as the physicochemicalproperties of pseudoazurin, were analyzed. All of the mutatedpseudoazurins showed optical spectra and oxidation-reductionpotentials almost identical to those of wildtype pseudoazurin,suggesting that none of the replacements of these lysine residuesaffected the environment around the type I copper site. Kineticanalysis of electron transfer between mutated pseudoazurinsand NIR reveals that the lysine mutations have very little effecton the rate of electron transfer to NIR, but substitution atresidues 10, 38, 57 and 77, all close to the copper site, substantiallydecreases the affinity of pseudoazurin for NIR. This suggeststhat pseudoazurin interacts with NIR through the region closeto the type I copper site. The refined X-ray structures of Lys38Aspand Lys10Asp/Lys38Asp show that the molecular structure hasindeed changed little. A new space group is observed for theLys109Ala mutant crystal. Crystal packing interactions changefor the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Aspand Lys59Ala mutants.