Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells

We have designed expression constructs containing sequencescorresponding to apolipoprotein(a) kringle FV types 1, 2 and10 and used these constructs to transfect human embryonic kidneycells. We have also expressed a mutant form of kringle FV type2 in which the N-linked glycosylation site has been removedby replacement of an asparagine residue with an alanine. Immunoprecipitationanalysis of [³⁵S]Cys-labeled transfected cell culture supernatantsresulted in the observation of two bands for kringle IV type1 (M_r 30 000 and 26 000), two bands for kringle IV type 2 (M_r25 000 and 22 000), two bands for kringle IV type 10 (M_r 27000 and 23 000) and one band for the glycosylation mutant (M_r22 000). In all cases, observed molecular weights greatly exceededthose predicted from amino acid sequence, suggesting the presenceof both N- and O-linked glycans. None of the recombinant singlekringles were observed to bind to fibrinogen as determined byELISA or by co-immunoprecipitation in the case of kringle IVtype 10 and only kringle IV type 10 was able to bind to lysine-Sepharose.These data suggest that apo(a) binding to fibrinogen/fibrinmay require motif(s) in addition to apo(a) kringle IV type 10.     

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogenactivator (t-PA) is in part associated with the presence ofthe kringle 2 domain in t-PA. Within this kringle 2 domain alysyl-binding site has been described. The plasminogen to plasminconversion by urokinase-type plasminogen activator (u-PA), incontrast to that of t-PA, is not enhanced in the presence offibrin. Within the u-PA kringle domain no lysyl-binding siteis found. To study whether introduction of a lysyl-binding sitein the u-PA kringle domain will make u-PA a fibrin-dependentplasminogen activator, three stretches of amino acid residuesof the u-PA kringle domain (A²⁸-Q³³, D⁵⁵-N⁵⁷ and G⁶⁷-V⁷²) weresubstituted by three stretches of amino acids from the correspondingpositions of the kringle 2 domain of t-PA (M²⁸-K³³, D⁵⁵-D⁵⁷and N⁶⁷-W⁷²). These changes resulted in the creation of thelysyl-binding site consensus of the kringle 2 domain (K³³, D⁵⁵,D⁵⁷, W⁶² and W⁷²) in the u-PA kringle. However, the resultingu-PA mutant did not interact with lysyl-Sepharose, nor did itdisplay fibrin-enhanced plasminogen activation in the presenceof soluble fibrin mimic. When the kringle domain of u-PA wasreplaced by the kringle 2 domain of t-PA, similar results wereobtained. The hybrid protein hardly interacted with lysyl-Sepharoseand the plasminogen activation was not enhanced in the presenceof fibrin mimic However, the N-terminal fragment isolated fromthis hybrid molecule (consisting of growth factor domain andkringle 2 domain) did interact with lysyl-Sepharose, suggestingthat in the hybrid molecule a functional lysyl-binding siteis present but not operational. Indeed, lysine analogue (e-amino-caproicacid) sensitive binding of isolated t-PA kringle 2 domain tou-PA could be observed. The modified u-PA kringle, the wildtype u-PA kringle and the kringle 2 of the u-PA hybrid werealso placed N-terminal of the protease domain of t-PA. As expected,the t-PA mutant consisting of the kringle 2 domain and the proteasedomain bound to lysyl-Sepharose and showed fibrin-dependentplasminogen activation. Further, the hybrid molecule consistingof the u-PA kringle placed N-terminal of the t-PA protease domaindid not display these features. Introduction of the modifiedu-PA kringle N-terminal of the t-PA protease domain resultedin a very weak interaction with lysyl-Sepharose. Despite thehigh overall similarity in primary structure of the modifiedu-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogenactivation of this hybrid molecule was observed. The above-mentionedresults question the concept that the structural auto-nomousdomains within hybrid plasminogen activators t-PA and u-PA functionas autonomous domains and suggest that interactions betweenthe kringle and the protease domain in hybrid molecules stronglyinfluences their functional features     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

The X-ray structure of (MeBm2t)1-cyclosporin complexed with cyclophilin A provides an explanation for its anomalously high immunosuppressive activity

For most of the cyclosporin A (CsA) analogs, there is generallya good correlation between cyclophilin binding and immunosuppression.However, this relationship does not seem to hold for 4-[(E)-2-butenyl]-4,4,N-trimethyl-L-threonine¹(MeBm₂t)¹-CsA.Its affinity for cyclophilin was reported to be {small tilde}1percent; that of CsA and its immunosuppressive activity invitro was shown to be {small tilde} 30% that of CsA. We reporthere the crystal structure of a complex between recombinanthuman cyclophilin A (CypA) and (MeBm₂t)¹-CsA which has beendetermined by X-ray crystallography at 2.2 Å resolutionand refined to an Rfactor of 16.3%. (MeBm₂t)¹-CsA shows a similarbound conformation and network of interactions to CypA as CsA.The measured lower affinity for CypA cannot therefore be explainedby a different mode of binding. We propose that the poor affinityto CypA could be accounted for by the existence of an equilibriumin aqueous solution between a ‘cyclophilin bound conformation’and a ‘nonbinding conformation’ of (MeBm₂t)¹-CsA.The relatively high immunosuppressive activity is suggestedto result from slight conformational differences observed inthe effector domain     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDS–PAGE ‘snapshot’ analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZ–GST::rFosLZ–GSTheterodimers whereas rJunLZ–GST::rFosLZ and rJunLZ::rFosLZ–GSTformed readily. Furthermore, rJunLZ–GST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (K_a >10⁶ M^-1) with no higher multimeric forms. rFosLZ–GST weaklyassociates beyond a dimer (K_a {small tilde}4x10⁵ M^-1) and rJunLZ–GSTassociates indefinitely (K_a {small tilde}4x10⁶ M^-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions

In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')₂ fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x10^–6 M for TNP and 5 x 10^–6 M for IgG F(ab')₂ fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')₂ fragments     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants

We have constructed an expression vector that leads to secretionof the whole Fc of human immunoglobulin E (hIgE-Fc) from mammaliancells at levels up to 100 mg/l of culture. Two surface glycosylationsites at Asn265 and Asn371 have been changed to glutamine, toobtain a more homogeneous preparation of hIgE-Fc for structuralstudies. Comparison of wild-type and mutant products revealedthat Asn371 is rarely glycosylated in Chinese hamster ovarycells. Both the double mutant and wild-type hIgEFc bind to thehigh-affinity IgE receptor, FcRI, with about the same affinityas myeloma IgE (K_a in the range 10¹⁰–10¹¹ M^–1),and were able to sensitize isolated human basophils for anti-IgEtriggering of histamine release. However, only the double mutanthIgE-Fc approached the affinity of myeloma IgE for the low-affinityreceptor, FcRII (K_a = 7.3x10⁷ M^–1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (K_a = 4.1x10⁶M^–1).     

Targeting myeloid leukemia with a DT390-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice

The IL-3 receptor was expressed on a high frequency of myeloidleukemia cells and also on hematopoietic and vascular cells.We previously showed that a recombinant IL-3 fusion immunotoxin(DT₃₉₀IL-3) expressed by splicing the murine IL-3 gene to atruncated diphtheria toxin (DT₃₉₀) gene selectively killed IL-3R⁺expressing cells and was not uniformly toxic to uncommited BMprogenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J.and Vallera,D.A., 1996, Blood, 88, 1445–1456). Thus, weexplored the feasability of using DT₃₉₀IL-3 as an anti-leukemiaagent. DT₃₉₀IL-3 was toxic when administered to mice at dosesas low as 0.1 µg/day. The dose limiting toxicity appearedto be related to platelet and bleeding effects of the fusiontoxin. Because of these effects, DT₃₉₀IL-3 was studied ex vivoas a means of purging contaminating leukemia cells from BM graftsin a murine autologous BM transplantation. In this setting,as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid32Dp210 leukemia cells were lethal. An optimal purging intervalof 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BMmixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneicmice. Mice given treated grafts containing BM and a lethal doseof 32Dp210 cells survived over 100 days while mice given untreatedgrafts did not survive (P < 0.00001). DT₃₉₀IL-3 may provehighly useful for ex vivo purging of lethal malignant leukemiacells from autologous BM grafts.     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain

A mutant of papain, where an inter-domain hydrogen bond betweenthe side chain hydroxyl group of a serine residue at position176 and the side chain carbonyl oxygen of a glutamine residueat position 19 has been removed by site-directed mutagenesis,has been produced and characterized kinetically. The mutationof Ser176 to an alanine has only a small effect on the kineticparameters, the k_cat/K_m for hydrolysis of CBZ-Phe-Arg-MCA bythe Serl76Ala enzyme being of 8.1 x 10⁴ /M/s compared with 1.2x 10⁵ /M/s for papain. Serine 176 is therefore not essentialfor the catalytic functioning of papain, even though this residueis conserved in all cysteine proteases sequenced. The pH-activityprofiles were shown to be narrower in the mutant enzyme by upto 1 pH unit at high ionic strength. This result is interpretedto indicate that replacing Ser 176 by an alanine destabilizesthe thiolate—imidazolium form of the catalytic site Cys25-Hisl59residues of papain. Possible explanations for that effect aregiven and the role of a serine residue at position 176 in papainis discussed.     

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.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.     

Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate

A chemically synthesized DNA linker coding for a peptide fragmentthat contains four histidines was fused in-frame to the 5'-endof the Bacillus stearothermophilus lactate dehydrogenase gene.The gene product, His₄/lactate dehydrogenase, could be purifiedto homogeneity using either immobilized metal (Zn²⁺)-affinitychromatography or affinity chromatography on oxamate agarose.The stability against heat and urea for the modified enzymeswas decreased as compared to the native lactate dehydrogenasebut could be increased if zinc ions were present during thedenaturation. In the presence of zinc ions the His₄/lactatedehydrogenase could catalyse the sequential reaction from oxaloacetateto L-lactate, hence operating as a semi-synthetic bifunctionalenzyme. A small increase in the apparent secondorder rate constant(k_cat/K_m) of the coupled reaction was observed as compared toa corresponding system with native lactate dehydrogenase.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.     

Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important forcatalysis are poorly understood. We have probed the functionof interdomain interactions by generating tPA variants in whichdomains are duplicated or rearranged. The proteins were expressedin a transient mammalian expression system and tested in vitrofor their ability to activate plasminogen, induce fibrinolysisand bind to a forming fibrin clot. Duplication of the heavychain domains of tPA produced enzymatically active tPA variants,many of which demonstrated similar in vitro amidolytic and fibrinolyticactivity and similar fibrin affinity to the parent molecule.Zymographic analysis of the domain duplication tPA variantsshowed one major active species for each variant. Selectionof the residues duplicated and the interdomain spacing werefound to be critical considerations in the design of tPA variantswith duplicated domains. We also rearranged the domains of tPAsuch that kringle 1 replaced the second kringle domain and viceversa. An analysis of these variants indicates that the firstkringle domain can confer fibrin affinity to a tPA variant andfunction in place of kringle 2. Therefore, in wild-type tPA,the functions of kringle 1 and kringle 2 must be dependent partiallyon their orientation within the heavy chain of the protein.The functional autonomy of the heavy and light chains of tPAis demonstrated by the activity of a tPA variant in which theorder of the heavy and light chains was reversed.     

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display

The catalytic histidine of human neutrophil elastase was replacedwith alanine (H57A) to determine if a substrate histidine couldsubstitute for the missing catalytic group—`substrate-assistedcatalysis'. H57A and wild-type elastase were recovered directlyfrom Pichia pastoris following expression from a synthetic genelacking the elastase pro sequence, thereby obviating the needfor zymogen activation. Potential histidine-containing substratesfor H57A elastase were identified from a phage library of randomizedsequences. One such sequence, REHVVY, was cleaved by H57A elastasewith a catalytic efficiency, k_cat/K_M, of 2800 s^–1 M^–1,that is within 160-fold of wild-type elastase. In contrast,wild-type but not H57A elastase cleaved the related non-histidinecontaining sequence, REAVVY. Ten different histidine-containinglinkers were cleaved by H57A elastase. In addition to the requirementfor a P2 histidine, significant preferences were observed atother subsites including valine or threonine at P1, and methionineor arginine at P4. A designed sequence, MEHVVY, containing thepreferred residues identified at each subsite proved to be amore favorable substrate than any of the phage-derived sequences.Extension of substrate-assisted catalysis to elastase suggeststhat this engineering strategy may be widely applicable to otherserine proteases thereby creating a family of highly specifichistidine-dependant proteases.