Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization.

Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme from bacteriophage T4 were used to explore the feasibility of using site-directed mutagenesis to generate isomorphous heavy-atom derivatives for protein crystallography. Cysteines 54 and 97, present in wild-type lysozyme, can be readily reacted with mercuric ion to produce an excellent isomorphous heavy-atom derivative. Mutants with an additional cysteine at position 86, 146, 153 or 157, or with Cys 97 replaced by Val, were engineered by site-directed mutagenesis. The mutant lysozyme Thr 157----Cys reacts with mercuric chloride to give an excellent new derivative although Cys 157 is only approximately 60% substituted with the heavy atom. The cysteine at position 146 is largely buried but reacts readily with mercuric chloride. In this case the isomorphism is poor and the resultant derivative is of marginal quality. Cys 153 reacts rapidly with mercuric ion but the derivative crystals do not diffract. The mutant Pro 86----Cys does not yield a particularly good heavy-atom derivative. This is due in part to a loss of isomorphism associated with the mutation. In addition, Cys 86 shows very little reactivity towards mercurials even though it is fully exposed to solvent. The mutation Cys 97----Val was used to explore the possibility of creating an independent derivative by deleting a heavy-atom site already present in wild-type lysozyme. In all cases that were tested, the quality of the heavy-atom derivative was improved by using as an isomorphous pair mercury-substituted mutant versus non-substituted mutant rather than mercury-substituted mutant versus (non-substituted) wild-type lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutant acyl-coenzyme A:cholesterol acyltransferase 1 devoid of cysteine residues remains catalytically active.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in the early stages of atherosclerosis. ACAT1 is an integral membrane protein with multiple transmembrane domains. Human ACAT1 contains nine cysteine residues; its activity is severely inhibited by various thiol-specific modification reagents including p-chloromercuribenzene sulfonic acid, suggesting that certain cysteine residue(s) might be near or at the active site. We constructed various ACAT1 mutants that contained either single cysteine to alanine substitution at various positions, contained a reduced number of cysteines, or contained no cysteine at all. Each of these mutants retained 20% or more of the wild-type ACAT activity. Therefore, cysteine is not essential for ACAT catalysis. For the cysteine-free enzyme, its basic kinetic properties and intracellular localization in Chinese hamster ovary cells were shown to be very similar to those of the wild-type enzyme. The availability of the cysteine-free ACAT1 will facilitate future ACAT structure function studies. Additional studies show that Cys467 is one of the major target sites that leads to p-chloromercuribenzene sulfonic acid-mediated ACAT1 inactivation, suggesting that Cys467 may be near the ACAT active site(s).     

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.     

Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes

Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.     

Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation

Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by introducing single mutant cysteine residues that were then thiol-specifically derivatized with the environmentally sensitive fluorophoracrylodan. Membrane binding of the labeled proteins was accompanied by spectral shifts of the probe fluorescence, suggesting that the toxin molecule had undergone a conformational change. Here we provide evidence that this change corresponds to an allosteric transition of the toxin monomer that is required for the subsequent oligomerization and pore formation. The conformational change is reversible with reversal of binding, and it is related to temperature in a fashion that closely parallels the temperature-dependency of oligomerization. Furthermore, we describe a point mutation (N402E) that, while compatible with membrane binding, abrogates the accompanying conformational change. At the same time, the N402E mutation also abolishes oligomerization. These findings corroborate the contention that the target membrane acts as an allosteric effector to activate the oligomerizing and pore-forming capability of streptolysin O.     

Cholesterol microcrystals and cochleate cylinders: Attachment of pyolysin oligomers and domain 4

Using an established organic solvent injection procedure for the preparation of aqueous cholesterol microcrystal suspensions, it has now been shown that a new, hollow, cylindrical, tightly-coiled, multi-bilayer form of cholesterol can be generated, termed the cochleate cylinder. Cholesterol cochleate cylinders are formed in larger numbers at intermediate temperatures (40–75 °C) but are not formed at 100 °C. The structure of the cholesterol microcrystals and cochleate cylinders is shown in negatively stained electron micrographs. Oligomerization and attachment of pyolysin to cholesterol microcrystals and cochleate cylinders is shown, as is the attachment of the pyolysin “cholesterol-binding” domain 4 (D4) fragment. The bound D4 domain forms a linear array on the two planar surfaces and edges of the cholesterol microcrystals and a quasi helical array on the surface of the cochleate cylinders. Little evidence has been obtained to support the possibility that interaction or hetero-oligomerization can occur between intact pyolysin and the pyolysin D4 fragment on the surface of cholesterol microcrystals. Using immobilized cholesterol crystals attached to a carbon support film, single-sided linear labelling of the cholesterol surface with pyolysin D4 has been achieved, which correlates well with the images from the microcrystal suspensions and our earlier data using non-cytolytic streptolysin O mutants.     

Structural and functional consequences of inactivation of human glutathione S-transferase P1-1 mediated by the catechol metabolite of equine estrogens, 4-hydroxyequilenin

The inactivation mechanism(s) of human glutathione S-transferase P1-1 (hGST P1-1) by the catechol metabolite of Premarin estrogens, 4-hydroxyequilenin (4-OHEN), was (were) studied by means of site-directed mutagenesis, electrospray ionization mass spectrometric analysis, titration of free thiol groups, kinetic studies of irreversible inhibition, and analysis of band patterns on nonreducing sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The four cysteines (Cys 14, Cys 47, Cys 101, and Cys 169 in the primary sequence) in hGST P1-1 are susceptible to electrophilic attack and/or oxidative damage leading to loss of enzymatic activity. To investigate the role of cysteine residues in the 4-OHEN-mediated inactivation of this enzyme, one or a combination of cysteine residues was replaced by alanine residues (C47A, C101A, C47A/C101A, C14A/C47A/C101A, and C47A/C101A/C169A mutants). Mutation of Cys 47 decreased the affinity for the substrate GSH but not for the cosubstrate 1-chloro-2,4-dinitrobenzene (CDNB). However, the Cys 47 mutation did not significantly affect the rate of catalysis since V(max) values of the mutants were similar or higher compared to that of wild type. Electrospray ionization mass spectrometric analyses of wild-type and mutant enzymes treated with 4-OHEN showed that a single molecule of 4-OHEN-o-quinone attached to the proteins, with the exception of the C14A/C47A/C101A mutant where no covalent adduct was detected. 4-OHEN also caused oxidative damage as demonstrated by the appearance of disulfide-bonded species on nonreducing SDS--PAGE and protection of 4-OHEN-mediated enzyme inhibition by free radical scavengers. The studies of thiol group titration and irreversible kinetic experiments indicated that the different cysteines have distinct reactivity for 4-OHEN; Cys 47 was the most reactive thiol group whereas Cys 169 was resistant to modification. These results demonstrate that hGST P1-1 is inactivated by 4-OHEN through two possible mechanisms: (1) covalent modification of cysteine residues and (2) oxidative damage leading to proteins inactivated by disulfide bond formation.     

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.     

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.     

Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase

The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.     

Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria

Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.     

Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis.

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-CTX sensitivity (IC50 = 1.9 +/- 0.1, 4.9 +/- 0.9, and 5.5 +/- 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan-to-cysteine mutants partially or fully restored the wild-type mu-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.     

Evidence for catalytic cysteine-histidine dyad in chalcone synthase

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin.

The roles of selected amino acid residues of human 14-kDa beta-galactoside-binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate beta-galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.     

Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore. Received: 18 May 1999/Revised: 18 September 1999     

Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.