Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

Recombinant pro-regions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes

Proteolytic enzymes require the presence of their proregionsfor correct folding. Of the four proteolytic enzymes from Caricapapaya, papain and papaya proteinase IV (PPIV) have 68% sequenceidentity. We find that their proregions are even more similar,exhibiting 73.6% identity. cDNAs encoding the pro-regions ofthese two proteinases have been expressed in Escherichia coliindependently from their mature enzymes. The recombinant pro-regionsof papain and PPIV have been shown to be high affinity inhibitorsof all four of the mature native papaya cysteine proteinases.Their inhibition constants are in the range 10^–6–;10^–;9M. PPIV was inhibited two to three orders of magnitude lesseffectively than papain, chymopapain and caricain. The pro-regionof PPIV, however, inhibited its own mature enzyme more effectivelythan did the proregion of papain. Alignment of the sequencesof the four papaya enzymes shows that there is a highly variablesection towards the C-terminal of the pro-region. This regionmay therefore confer selectivity to the pro-regions for theindividual proteolytic enzymes.     

Mutational analysis supports a structural model for the cell cycle protein kinase p34

Structural models for the eukaryotic cell cycle control proteinp34 from human, S.pombe and S.cerevisiae have been derived fromthe crystallographic coordinates of the cAMP-dependent proteinkinase (cAPK) catalytic subunit (active conformation) and comparedwith the structure of Inactive CDK2 apoenzyme. Differences betweenthe p34 and cAPK catalytic sites provide a possible explanationfor their different substrate specificities. The p34 modelslocalize Tyrl5 and Thrl4 close to the sites of catalysis andsubstrate recognition where their phosphorylatlon could inhibitp34 kinase activity either by blocking MgATP or substrate binding.The conserved sequences PSTAIRE and LYLIFEFL are both closeto the catalytic site and accessible on the protein surfaceavailable to mediate interactions with other proteins. It ispredicted that p34 has an active-site cleft composed almostentirely of sequences common to all protein kinases and sequencesunique to the p34 protein family. Genetic and biochemical analysesof p34 have shown that it interacts extensively with a numberof other proteins. The model allows the relative dispositionof these sites of mutation to each other and to the sites ofcatalysis and substrate recognition to be appreciated. Surfaceregions on p34 that are important for function have been identified.These sites identify residues that may interact with p13^SUCL,cydin, plO7^WEEL and p80^cdc25     

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.     

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase

A mutant of Lactobacillus casei dihydrofolate reductase hasbeen constructed in which Thr63, a residue which interacts withthe 2'-phosphate group of the bound coenzyme, is replaced byalanine. This substitution does not affect k_cat, but producesan 800-fold increase in the K_m for NADPH, which reflects dissociationof NADPH from the enzyme-NADPH-tetrahydrofolate complex, anda 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociationconstant for the enzyme-NADPH complex. The difference in magnitudeof these effects indicates a small effect of the substitutionon the negative cooperativity between NADPH and tetrahydrofolate.Stopped-flow studies of the kinetics of NADPH binding show thatthe weaker binding arises predominantly from a decrease in theassociation rate constant. NMR spectroscopy was used to comparethe structures of the mutant and wild-type enzymes in solution,in their complexes with methotrexate and with methotrexate andNADPH. This showed that only minimal structural changes resultfrom the mutation; a total of 47 residues were monitored fromtheir resolved ¹H resonances, and of these nine in the binarycomplex and six in the ternary differed in chemical shift betweenmutant and wild-type enzyme. These affected residues are confinedto the immediate vicinity of residue 63. There is a substantialdifference in the ³¹P chemical shift of the 2'-phosphate ofthe bound coenzyme, reflecting the loss of the interaction withthe side chain of Thr63. The only changes in nuclear Overhausereffects (NOEs) observed were decreases in the intensity of NOEsbetween protons of the adenine ring of the bound coenzyme andthe nearby residues Leu62 and Ile102, showing that the substitutionof Thr63 does cause a change in the position or orientationof the adenine ring in its binding site.     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser¹⁴⁸ – Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedP_i with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site P_s. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser¹⁴⁸, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser¹⁴⁸ with the C(2)-hydroxyl of thesubstrate. The changed K_m for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the P_i site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

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.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the aminoacids directly involved in chemical catalysis. They mainly actas a general acid–base, electrophilic or nucleophiliccatalyst or they polarize and stabilize the transition state.An analysis of the structural features of 36 catalytic residuesin 17 enzymes of known structure and with defined mechanismis reported. Residues that bind metal ions (Zn² and Cu²) areconsidered separately. The features examined are: residue type,location in secondary structure, separation between the residues,accessibility to solvent, intra-protein electrostatic interactions,mobility as evaluated from crystallographic temperature factors,polarity of the environment and the sequence conservation betweenhomologous enzymes of residues that were sequentially or spatiallyclose to the catalytic residue. In general the environment ofcatalytic residues is similar to that of polar side chains thathave low accessibility to solvent. Two algorithms have beendeveloped to identify probable catalytic residues. Scanningan alignment of homologous enzyme sequences for peaks of sequenceconservation identifies 13 out of the 16 catalytic residueswith 50 residues overpredicted. When the conservation of thespatially close residues is used instead, a different set of13 residues are identified with 47 residues overpredicted. Acombination of the two algorithms identifies 11 residues with36 residues overpredicted.     

Selective cysteine <- serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens containsfive sulfhydryl groups per subunit. Cysteine serine replacementsshow that the thiols are not essential for catalysis. The increaseddissociation constant for FAD in mutant Cysl58Ser suggests thatCysl58 is important for the solvation of the pyrophosphate moietyof the prosthetic group. Wild-type p-hydroxybenzoate hydroxylaseis rapidly inactivated by mercurial compounds. Inactivationby a spin-labeled derivative of p-chloromercuribenzoate is fullyabolished in mutant Cys211Ser. Incorporation of the spin labelin the other Cys Ser mutants strongly impairs substrate bindingwithout affecting the catalytic properties of the FAD. The resultsare discussed with respect to previous tentative assignmentsfrom chemical modification studies and in light of the 3-D structureof the enzyme-substrate complex.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

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.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Comparative molecular modeling of Amphioxus calcium vector protein with calmodulin and troponin C

Calcium vector protein (CaVP), a new protein isolated from Amphioxusmuscle, binds in a Ca²⁺ -rgulated manner to a 27 kd target protein,named CaVPT, whose function has not been elucidated yet. CaVPbears significant sequence homology to both calmodulin and skeletalmuscle troponin C, especially in the C-tenninal half of themolecule, which presumably contains the two functional Ca²⁺sites. The N-terminal half contains two abortive EF-hands andis intramolecularly crosslinked with a disulfide bond. Usingthe crystallographic structures of calmodulin and striated muscletroponin C as a framework, we constructed two different three-dimensionalmodels of CaVP and modeled the intramolecular disulfide bridge.The modeling based upon the coordinates of calmodulin yieldsa Ca²⁺ -filled sites configuration in the N-terminal half ofthe molecule, even though no Ca²⁺ is bound in this half, whereasthe troponin C-derived model generates a Ca²⁺ -empty sites configuration.The models predict that neither in the Ca²⁺ nor in the Ca²⁺-empty sites conformation is there any steric and/or energeticobstacle for the formation of the disulfide bridge and thatthe disulfide bond is poorly accessible to reducing reagents.The optical properties of the Trp and Tyr residues of CaVP indicatethat the calmodulin-derived model represents the most plausibleprediction.     

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.     

Protein engineering of cytochrome c by semisynthesis: substitutions at Glutamic acid 66

We have used protein semisynthesis to prepare four analoguesof horse cytochrome c, in which the glutamic acid residue atposition 66 has been removed and replaced by norvaline, glutamine,lysine and, as a methodological control, glutamic acid. Thisresidue is quite strongly conserved in mitochondrial cytochromec, and forms part of a cluster of acidic residues that occursin all cytochromes c but whose function is obscure. Comparativestudies of the physical and biochemical properties of the analogueshave now disclosed two specific roles for Glu⁶⁶ in the protein.It contributes significantly to the stabilization of the activeconformation of the protein, probably by salt bridge formation,and it appears to participate in the redox-state-dependent ATP-bindingsite of cytochrome c. Our results also support two general viewsof the role of surface charged residues in cytochrome c, namelythat their disposition influences both redox potential, throughthe electrostatic field felt at the redox centre, and the kineticsof electron transfer, through the dipole moment they generate.     

Engineering of the substrate-binding region of the sublilisin-like, cell-envelop proteinase of Lactococcus lactis

The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide _g1-casein(l–23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137–139AKTalong one side of the binding cleft produced the 137–139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137–139GLA mutant with increased activity andbroader specificity. Furthermore, the 137–139GDT mutanthad a specificity towards _g1,-casein(l–23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis.     

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.