Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator

The tetra-alanine substitution variant KHRR 296–299 AAAAof tissue-type plasminogen activator (t-PA) was previously shownto have enhanced fibrin specificity and enhanced activity inthe presence of fibrin compared with the wild-type form of themolecule. The structural requirements for these alterationsin enzymatic activity were investigated by constructing severalamino acid substitution variants at each of the positions from296 to 299 and evaluating their activities under a variety ofconditions. Effects on plasminogen activator activity were commonamong the point mutants at positions 296–299; nearly allhad a phenotype similar to the KHRR 296–299 AAAA variant.The greatest effects on enzymatic function were found with multiplesubstitution variants, but some single charge reversals andproline substitutions had substantial effects. The enhancedfibrin specificity of KHRR 296–299 AAAA t-PA results inless fibrinogenolysis than seen with wild-type t-PA. Approximatelyfour times greater concentration of KHRR 296–299 AAAAcompared with wild-type t-PA was required to consume 50% ofthe fibrinogen in human plasma.     

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     

Locating the unpaired cysteine of tissue-type plasminogen activator

Variants of tissue-type plasminogen activator (t-PA) were constructedwith selected cysteines replaced by alanine to evaluate therole of an unpaired cysteine, which has been presumed to bein the growth factor module. C75A, C83A, C84A and CC83–84AAvariants of t-PA were expressed transiently in human embryonickidney cells. The biochemical properties of these variants providedexperimental evidence to identify the unpaired cysteine in t-PA.Assays of amidolytic activity, plasminogen activation (in thepresence or absence of fibrinogen or fibrin), plasma clot lysis,fibrin binding, clearance in mice, and interaction with a panelof monoclonal antibodies were performed as the basis for comparingthese variants with wild-type t-PA. In all assays, C83A t-PAwas biochemically equivalent to wild-type t-PA. C75A t-PA, C84At-PA and CC83-84AA t-PA variants exhibited reduced activitiesin a variety of functional assays. These variants displayedtwo- to threefold lower activity in fibrinogen or fibrin stimulatedplasminogen activation, and fivefold reduced plasma clot lysisactivity compared with that of wild-type t-PA. The affinityof C75A t-PA and C84A t-PA for fibrin was decreased more thantwo orders of magnitude compared with C83A t-PA or wild-typet-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced2-fold in mice. The C75A, C84A and CC83–84AA variantsdisplayed significantly decreased reactivity with anti-tPA monoclonalantibodies specific for finger/growth factor domain epitopes.These data are consistent with a disulfide linkage of Cys75with Cys84 and that Cys83 exists as an unpaired sulfhydryl.The significance of the unpaired cysteine is as yet undeterminedsince C83A t-PA and wild-type t-PA are functionally equivalent.     

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.     

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare the variants with linker-inserts placed at the BglII(115)and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively),which encode insertions at the boundaries of the finger domain.The variants with linker-inserts in the light chain (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.     

The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371–377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 23–47%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 20–70% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-typeplasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimideleads to a decrease in affinity for lysine and fibrin, to adecrease of plasminogen activation activity in the presenceof a fibrin mimic, but leaves amidolytic activity and plasminogenactivation without fibrin mimic unaffected. Experiments withkringle-2 ligands and a deletion mutant of t-PA (K₂P) suggeststhat glutamic or aspartic acid residues in K₂ of t-PA are involvedin stimulation of activity, lysine binding and fibrin binding.Mutant t-PA molecules were constructed by site-directed mutagenesisin which one or two of the five aspartic or glutamic acid residuesin K₂ were changed to asparagine or glutamine respectively.Mutation of Asp236 and/or Asp238 leads to t-PA molecules with3- to 4-fold lower specific activity in the presence of fibrinmimic and having no detectable affinity for lysine analogs.However, fibrin binding was not influenced. Mutation of Glu254also leads to a 3- to 4-fold lower activity, but to a much smallerreduction of lysine or fibrin binding. Residues Asp236 and Asp238are both essential for binding to lysine derivatives, whileGlu254 might be involved but is not essential. Residues Asp236,Asp238 and Glu254 are all three involved in stimulation of activity.Remarkably, mutation of residues Asp236 and/or Asp238 appearsnot to influence fibrin binding of t-PA whereas that of Glu254does.     

Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins

We have designed and constructed a DNA sequence encoding humantissue plasminogen activator (tPA) with convenient restrictionsites that flank each of the domains of the heavy chain. Toaccomplish this, the first 1095 bases of the gene coding forthe mature protein were synthesized with unique restrictionsites engineered into the interdomainal regions. This syntheticconstruction was then ligated to a cDNA fragment of the tPAgene that encoded the active site, thus generating a full-lengthtPA gene. The gene products produced by Chinese hamster ovary(CHO) cells transfected with either the tPA cassette gene orthe tPA cDNA gene were then compared with the tPA produced byBowes melanoma cells to determine whether or not synthetic interdomainalamino acid changes had an effect on the biochemical characteristicsof the molecule. Specifically, molecular weight, specific activity,enhancement by fibrinogen fragments and kinetic constants wereanalysed. None of the properties examined were significantlydifferent from those of the native melanoma tPA. Therefore,the cassette gene described herein should provide considerableversatility and precision in the construction of tPA mutantsby facilitating the manipulation of the finger, growth factorand kringle domains, and likewise should be useful in assessingthe function of these domains within the tPA molecule. We presentthis cassette gene system as a model for the analysis of proteindomain function applicable to other multi-domain proteins.     

Mutations affecting the activity of urokinase-type plasminogen activator

Mutagenesis throughout the single-chain urokinase-type plasminogenactivator (scu-PA) cDNA molecule, followed by expression ofthe mutant genes and secretion of the resulting mutant proteinsfrom yeast, has been used to determine the amino acid residuesimportant for activity of scu-PA molecules. Twelve out of 13colonies secreting variant scu-PA molecules with decreased abilityto form a zone of fibrinolysis had mutant genes with a singlecodon alteration in the serine protease encoding domain (B-chain).Many of these changes are of highly conserved residues in theserine proteases and are consequently of considerable interest.A model three-dimensional structure of the protease domain ofurokinase was used to explain the basis for the effects of thesedown mutations. The model showed that the strongest down mutationsresult from either interference of the mutated side chain withsubstrate binding at the active site or the introduction ofbulky or charged groups at structurally sensitive internal positionsin the molecule. Attempts to find second site revertants offive down mutants, altered either at the plasmin activationsite or near the serine at the active site, only resulted insame-site revertants, with the original or closely related aminoacids restored.     

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.     

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono-and diglyceride specific lipase

The extracellular lipase from Penicillium camembertii has uniquesubstrate specificity restricted to mono- and diglycerides.The enzyme is a member of a homologous family of lipases fromfilamentous fungi. Four of these proteins, from the fungi Rhizomucormiehei, Humicola lanuginosa, Rhizopus delemar and P.camembertii,have had their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymes exhibitsignificant differences. For example, the thermo-stability ofthe P.camembertii lipase is considerably lower than that ofthe H.lanuginosa enzyme. Since only the P.camembertii enzymelacks the characteristic long disulfide bridge, correspondingto Cys22–Cys268 in the H.lanuginosa lipase, we have engineeredthis disulfide into the former enzyme in the hope of obtaininga significantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety of biophysicaltechniques. The extra disulfide link was found to increase themelting temperature of the protein from 51 to 63°C. However,no difference is observed under reducing conditions, indicatingan intrinsic instability of the new disulfide. The optimal temperaturefor catalytic activity decreased by 10°C and the optimumpH was shifted by 0.7 units to more acidic.     

Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine

The method of Smith and Hartman [J. Biol. Chem., 263, 4921-4925(1988)] for introducing the non-natural lysine analog, S-(2-aminoethyl)cysteine,into specific sites in proteins by alkylation of a geneticallyintroduced cysteine with 2-bromoethylamine has been generalizedto be applicable to proteins containing one or more endogenouscysteines. The target cysteine residue introduced at the activesite of aspartate aminotransferase is protected by bound cofactor.The enzyme is partially unfolded in low concentrations of urea,and the non-active site cysteine residues derivatized by a reversiblethiol protecting reagent. The active site cysteine is then exposedand alkylated in 6 M urea. Enzyme activity is regenerated byremoval of the thiol protecting groups and refolding of theprotein.     

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential

The blood clotting enzyme thrombin plays a central role in theaetiology of occlusive disorders such as stroke and acute myocardialinfarction. During fibrinolytic therapy with plasminogen activators,thrombin is neutralized by anticoagulative drugs. In order tocombine plasminogen-activating and thrombin-inhibitory activitieswe constructed chimeric derivatives of recombinant single-chain,urokinase-type plasminogen activator (rscu-PA) which comprisethe kringle and protease domain of rscu-PA fused via a linkersequence to a thrombin-inhibitory domain. The inhibitory domaincontains a sequence element directed to the active site of thrombinand a sequence taken from either hirudin or the human thrombinreceptor both binding to the fibrinogen recognition site ofthrombin. Analysing different sets of point mutants showed thatthe linker between the protease domain and the active sitedirectedsequence is contributing significantly to the thrombin-inhibitorypotential. Kinetic analysis of thrombin inhibition revealedthat most of the chimeras tested competitively inhibit the thrombin-mediatedcleavage of a peptide substrate in a concentration-dependentmanner; however, in two examples the insertion of one glycineresidue into the active site directed-sequence abolished theblockade of the active site. This supports the conclusion thatthe chimeras with high thrombin-inhibitory potential interactwith the active site and the fibrinogen recognition site ofthrombin.     

Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain

The human urokinase plasminogen activator (uPA) epidermal growthfactor-like domain (residues 1-48) and a variant with a C-terminalepitope tag have been secreted from recombinant yeast Purifiedhuman uPA 1-48 and uPA l-48glu compete for binding to the humanuPA receptor with Kji of 180 and 400 pM respectively, in anin vitro assay using an immobilized recombinant uPA receptor.A synthetic gene encoding human uPA 1-48 with an N-terminalepitope tag was inserted into a phagemid expression vector asa fusion with residues 249-406 of the M13 pill protein withan intervening amber codon (TAG). Phagemid production led toinfectious particles which were selectively bound and elutedfrom both epitope tag antibody and urokinase receptor. Sequentialbinding to this antibody and receptor demonstrated a substantialenrichment, where up to 10% of the infectious particles werethen retained on urokinase receptor-coated plates. A PCR strategywas used to convert previously described peptide bacteriophageligands for the urokinase receptor to phagemid display. Theyields of these peptide phagemids and the uPA 1-48 phagemidshowed a correlation with peptide affinity, in contrast to whenthe peptides are multivalently displayed on a bacteriophage.     

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis

Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Gly^ß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Gly^ß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis inan attempt to increase the cytotoxic potency and receptor-bindingaffinity of the diphtheria-toxin-related interleukin-2 (IL-2)fusion toxins. Previous studies have demonstrated that boththe DAB₄₈₆-IL-Z and DAB₃₈₉-IL-2 forms of the fusion toxin consistof three functional domains: the N-tenninal fragment-A-assodatedADP-ribosyltransferase, the hydrophobk-membrane-associatingdomains, and the C-terminal receptor-binding domain of humanIL-2. By insertion mutagenesis we have increased the apparentflexibility of the polypeptide chain between the membraneassociatingdomains and the receptor-binding domain of this fusion toxin.In comparison to DAB₄₈₆-IL-2, the cytotoxic potency of the insertionmutants was increased by 17-fold for high-affinity IL-2-receptor-bearingcell lines in vitro. Moreover, competitive displacement experimentsusing [¹²⁵I]rIL-2 demonstrate that the increase in cytotoxicpotency correlates with an increase in receptor-binding affinityfor both the high and intermediate forms of the IL-2 receptor.     

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.     

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

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

Structural and functional domains in human tumour necrosis factors

Human tumour necrosis factors (hTNFs) and ß are relatedpleiotropic cytokines which share many activities and competewith each other for binding to two receptor components on manycell types. Although structural and biological data indicatethat the active form of hTNF- may be a symmetrical trimer, themanner in which hTNFs interact with their receptors to triggera myriad of cell type-dependent responses is not clear. A combinationof chemical modification, epitope mapping and site-directedmutagenesis approaches suggest that at least four distinct peptidesequences are Important for the biological activity of hTNF-.In particular, certain peptide sequences between amino acidpositions 11 and 35 in hTNF- appear to be critical for receptorbinding and triggering biological responses. The recent cloningof the two hTNF-/ß receptors opens the way for precisemapping of the functional domains in hTNFs