Binding of antimitotic drugs around cysteine residues of tubulin

Two independent approaches provide evidence of cysteine residues in the vicinity of the binding sites of colchicine and vinblastine to tubulin: (1) The reactive bromoacetamide group of the affinity label bromocolchicine covalently binds to cysteine residues of tubulin; (2) vinblastine and colchicine slow down the reaction of DTNB with SH groups of tubulin.     

Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding

Sulfhydryls and disulfides of platelet tubulin have been quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by two cycles of temperature-dependent polymerization--depolymerization was found to contain 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, one intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by one which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding, on the other hand, was only minimally influenced by blocking of sulfhydryls.     

The local electrostatic environment determines cysteine reactivity of tubulin

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide, N-[(14)C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.     

Autopalmitoylation of tubulin

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.     

Reactivities of sulfhydryl groups in native and metal-free aminoacylase I

Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.     

The action of cytochalasin A on the in vitro polymerization of brain tubulin and muscle G-actin

The presence of cytochalasin A inhibits the self-assembly of beef brain tubulin and rabbit muscle G-actin in vitro and also decreases the colchicine binding of tubulin. Prior reaction of cytochalasin A with 2-mercaptoethanol destroys its inhibitory effects. It is shown that cytochalasin A exerts its actions by reacting with sulfhydryl groups, possibly causing irreversible structural changes in the proteins. Cytochalasin B does not affect the tubulin assembly reaction.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

Excimer fluorescence of pyrene-maleimide-labeled tubulin.

Excimer-forming cysteines in tubulin are detected by the presence of excimer fluorescence in N-(1-pyrenyl)maleimide-labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N-(1-pyrenyl)maleimide-labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N-(1-pyrenyl)maleimide-labeled tubulin showed that the loss of excimer fluorescence precedes subunit dissociation. The loss of both colchicine-binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Sulfhydryls and the in vitro polymerization of tubulin

The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl- oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-A ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitol, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaCl2 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.     

Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro.

The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal tubulin.     

Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin.

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.     

Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Involvement of tryptophan residues in colchicine binding and the assembly of tubulin

Chemical modification of tubulin with 2-hydroxy-5-nitrobenzyl bromide, a reagent selective for tryptophan, inhibits tubulin's colchicine binding and in vitro assembly activities. Loss of colchicine binding shows a linear relationship with the modification of tryptophan residues, and is complete when not more than five residues are modified. GTP affords partial protection against this loss of colchicine binding. The in vitro assembly of tubulin is somewhat less sensitive, since microtubules are formed from tubulin dimers possessing 3–4 but not five modified residues. Furthermore, two of the eight tryptophans per dimer are reactive when tubulin is assembled into microtubules.     

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.     

Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites.

Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.     

Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.     

Cytochalasin A inhibits the in vitro polymerization of brain tubulin and muscle actin.

Cytochalasin A (CA) inhibits the self-assembly of beef brain tubulin. The concentrations necessary to cause the inhibition are only slightly higher than the tubulin concentration. Cytochalasin B (CB) at identical and higher concentrations has no noticeable effect. Cytochalasin A also inhibits colchicine binding activity suggesting that it denatures the tubulin molecule. The results indicate that the reaction of CA with the sulfhydryl groups of tubulin is responsible for its action. CA also prevents the conversion of G-actin to F-actin, probably via a similar mechanism.