Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.     

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

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

pH on-off switching of antibody-hapten binding by site-specific chemical modification of tyrosine

Tetranitromethane (TNM) chemically mutates the binding sitesof antibodies so that the nitrated antibodies exhibit pH-dependentbinding near physiological pH. Three monoclonal antibodies wereselectively modified, each under different conditions, withthe resultant loss of binding activity at pH > 8 which isrecovered at pH < 6. Recovery and loss of binding are ascribedto the protonation and deprotonation, respectively, of the hydroxylgroup of the resulting 3-nitrotyrosine side chain (pK_a 7) atthe binding site of these antibodies. pH on–off dependencyof binding activity, common to all TNM-modified antibodies studiedby us so far, may find use in a variety of applications in whichcontrolled modulation under mild conditions is required     

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.     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and itscomplexes with various species-variant and mutant lysozymeshave been the subject of considerable experimental and theoreticalinvestigation. The affinity of HyHEL-5 for bobwhite quail lysozyme(BWQL) is over 1000-fold lower than its affinity for the originalantigen, hen egg lysozyme (HEL). This difference is believedto arise almost entirely from the replacement in BWQL of thestructural and energetic epitope residue Arg68 by lysine. Inthis study, the association and dissociation kinetics of BWQLwith HyHEL-5 were investigated under a variety of conditionsand compared with previous results for HEL. HyHEL-5–BWQLassociation follows a bimolecular mechanism and the dissociationof the antibody–antigen complex is a first-order process.Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0to 10.0) produced about a 2-fold change in the association anddissociation rates. The effect of viscosity modifiers on theassociation reaction was also studied. The large differencein the HEL and BWQL affinities for HyHEL-5 is essentially dueto differences in the dissociation rate constant.     

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.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties

Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in k_cat but to no changein K_m, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected K_m a more than k_cat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased k_catonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect K_m. The Trpl78-Aspmutation greatly decreased k_cat while increasing K_m, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.     

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).     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

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.     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active siteof Escherichia coli alkaline phosphatase was investigated bysite-specific mutagenesis. A mutant version of alkaline phosphatasewas constructed with alanine in place of aspartic acid at position101. When kinetic measurements are carried out in the presenceof a phosphate acceptor, 1.0 M Tris, pH 8.0, both the k_cat andthe K_m, for the mutant enzyme increase by –2-fold, resultingin almost no change in the k_cat/K_m ratio. Under conditions ofno external phosphate acceptor and pH 8.0, both the k_cat andthe K_m for the mutant enzyme decrease by {small tilde}2-fold,again resulting in almost no change in the k_cat/K_m ratio. Thek_cat for the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-typeand mutant enzymes, as is the K₁ for inorganic phosphate. Thereplacement of aspartic acid 101 by alanine does have a significanteffect on the activity of the enzyme as a function of pH, especiallyin the presence of a phosphate acceptor. At pH 9.4 the mutantenzyme exhibits 3-fold higher activity than the wild-type. Themutant enzyme also exhibits a substantial decrease in thermalstability: it is half inactivated by treatment at 49°C for15 min compared to 71°C for the wild-type enzyme. The datareported here suggest that this amino acid substitution altersthe rates of steps after the formation of the phospho-enzymeintermediate. Analysis of the X-ray structure of the wild-typeenzyme indicates that the increase in catalytic rate of themutant enzyme in the presence of a phosphate acceptor may bedue to an increase in accessibility of the active site nearSerl02. The increased catalytic rate of this mutant enzyme maybe utilized to improve diagnostic tests that require alkalinephosphatase, and the reduced heat stability of the mutant enzymemay make it useful in recombinant DNA techniques that requirethe ability to heat-inactivate the enzyme after use.     

The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase

In previous kinetic studies of Escherichia coli aspartate aminotransferase,it was determined that some substitutions of conserved cysteine191, which is located outside of the active site, altered thekinetic parameters of the enzyme (Gloss,L.M., Spencer,D.E. andKirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195–208).The mutations resulted in an alkaline shift of 0.6–0.8pH units for the pK_a of the internal aldimine between the PLPcofactor and Lys258. The change in the pK_a affected the pH dependenceof the k_cat/K_m (aspartate) values for the mutant enzymes. Tohelp to understand these observations, crystal structures offive mutant forms of E.coli aspartate aminotransferase (themaleate complexes of C191S, C191F, C191Y and C191W, and C191Swithout maleate) were determined at about 2 Å resolutionin the presence of the pyridoxal phosphate cofactor. The overallthree-dimensional fold of each mutant enzyme is the same asthat of the wild-type protein, but there is a rotation of themutated side chain around its C     

A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs

Using a series of homologous calcium-binding proteins, a quantitativestructure–activity relationship (QSAR), log(1/K_d) = –18.986– 1.6278(X₁) + 0.7981(X₂) + 0.2312(X₃), has been established,which relates the calcium-binding affinities (1/K_d) of the regulatoryproteins with (i) the net ligand charge (X₁) of the two calciumbinding loops, (ii) the hydrophobicity (X₂) of the ß-sheetsegment of the loops and (iii) the hydrophobicity (X₃) of thefour ‘EF-hand’ helices. It is found that the bindingaffinities are influenced by the ‘EF-hand’ pairrather than the individual ‘EF-hands’. The QSAR,in addition to explaining satisfactorily the large variationin the observed calcium affinities, can predict the affinitiesof the ‘EF-hand’ pairs in other proteins from theamino acid sequence and can also account for the changes inthe affinities caused by substitution in the hydrophobic and/ormetal-coordinating residues. Thus, this relationship can beemployed in protein design and engineering. The method is potentiallyuseful in the development of similar relationships for the bindingof other proteins to substrates, inhibitors, drugs and co-factors.     

Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background

To change the substrate preference of carboxypeptidase Y theputative substrate binding pocket was subjected to random mutagenesis.Based upon the three-dimensional structure of a homologous enzymefrom wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216,Ile340 and Cys341 are the amino acid residues of carboxypeptidaseY that constitute S₁ the binding pocket for the penultimateamino acid side chain of the substrate. We developed a new andgenerally applicable mutagenesis strategy to facilitate efficientscreening of a large number of mutants with multiple changesin carboxypeptidase Y. The key feature is the elimination ofwild type background by introducing a nonsense codon at eachtarget site for subsequent mutagenesis by degenerate oligonucleotides.The entire hypothesized S₁ binding pocket and subsets of itwere subjected to saturation mutagenesis by this strategy, andscreening yielded a number of mutant enzymes which have up to150 times more activity (k_cat/K_m towards CBZ-LysLeu-OH thanthe wild type enzyme. All selected mutants with increased activityhave mutations at position 178. Mutagenesis of positions 215and 216 has virtually no effect on the activity, while mutatingpositions 340 and 341 generally reduces activity.     

Making tissue-type plasminogen activator more fibrin specific

The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296–299or DDD 364–366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466–470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe k_cat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296–299, where the presenceof fibrin manifests significant increases in both k_cat and K_m.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity.     

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.     

The sequence and X-ray structure of the trypsin from Fusarium oxysporum

The trypsin from Fusarium oxysporum is equally homologous totrypsins from Streptomyces griseus, Streptomyces erythraeusand to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibitedform of the enzyme has been crystallized from 1.4 M Na₂SO₄,buffered with citrate at pH 5.0–5.5. The crystals belongto space group P2₁ with cell parameters a=33.43 Å, b=67.65Å, c=39.85 Å and ß=107.6°. There isone protein molecule in the asymmetric unit. X-ray diffractiondata to a resolution of 1.8 Å were collected on film usingsynchrotron radiation. The structure was solved by molecularreplacement using models of bovine and S.griseus trypsins andrefined to an R-factor of 0.141. The overall fold is similarto other trypsins, with some insertions and deletions. Thereis no evidence of the divalent cation binding sites seen inother trypsins. The covalently bound inhibitor molecule is clearlyvisible.