The modification of cholinesterase activity by 5′,5′-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site

1. Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6'-dithiodinicotinic acid and 2,2'-dithiobis-(5-nitropyridine) less so and 2,2'-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5'-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the K(m) value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under V(max.) conditions in addition to K(m) being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.     

Porcine liver aminopeptidase. Further characterization of its sulfhydryl groups

Porcine liver aminopeptidase was inactivated by various sulfhydryl-reactive reagents, whose inactivation rates were in the order: p-chloromercuribenzoate(PCMB) greater than HgCl2 greater than 2,2'-dithiodipyridine greater than 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB). The processes of inactivation by these reagents did not follow pseudo-first-order kinetics, and prolonged incubation did not alter the level of maximum inactivation. The substrates provided no protection against the inactivation by DTNB, and the numbers of sulfhydryl groups titrated with the reagent were not influenced by the presence or absence of puromycin (a competitive inhibitor). The modification of sulfhydryl groups caused a slight increase in the Km value for the enzyme and a significant decrease of the Vmax value. There are two ionizable groups (pKe, 6.2; 7.8 and pKes, 6.0; 7.8) in the catalytic action of the enzyme. From the pKi vs. pH profile of inhibition with PCMB, the pK value of 7.8 does not correspond to the ionization of a sulfhydryl group. The thiol-modified enzyme was activated by cobalt ion, as was the native enzyme (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101). But in contrast with the native enzyme, the thiol-modified enzyme was activated about 2.5-fold and the maximum activation remained almost constant during prolonged incubation with cobalt ion. These results suggest that the sulfhydryl groups of the enzyme are located apart from the binding site of cobalt ion and do not participate directly in the catalytic process.     

Kinetics of inactivation of creatine kinase during modification of its thiol groups

Kinetics of inactivation and modification of the reactive thiol groups of creatine kinase by 5,5'-dithiobis(2-nitrobenzoic acid) or iodoacetamide have been compared, the former by following the substrate reaction in presence of the inactivator [Wang, Z.-X., & Tsou, C.-L. (1987) J. Theor. Biol. 127, 253]. The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. From the results obtained it appears that with respect to ATP both inactivators are noncompetitive whereas for creatine iodoacetamide is competitive but DTNB is not. The formation of the ternary complex protects against the inactivation by both DTNB and iodoacetamide. The inactivation kinetics is monophasic with both inactivators, but under similar conditions, the modification reactions in the presence of the transition-state analogue of creatine-ADP-Mg2+-nitrate show biphasic kinetics as also reported by Price and Hunter [Price, N.C., & Hunter, M.G. (1976) Biochim. Biophys. Acta 445, 364]. If the reactive ternary complex and the enzyme complexed with the transition-state analogue react in the same way with these reagents, the modification of one fast-reacting thiol group for each enzyme molecule leads to complete inactivation, indicating that the enzyme has to be in the dimeric state to be active.     

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

Differential response of cysteine residues in pig muscle phosphoglucose isomerase to seven sulfhydryl-modifying reagents

The three cysteine residues per subunit of pig muscle phosphoglucose isomerase show different reactivities toward various sulfhydryl reagents. The organomercurial, p-mercuribenzoate, can titrate two of the sulfhydryl groups under nondenaturing conditions. 2,2'-Dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, methyl 2-pyridyl disulfide, and 2-(2'-pyridylmercapto)mercuri-4-nitrophenol all label only one sulfhydryl group under the same conditions, whereas iodoacetic acid does not react with any of the sulfhydryl groups except when the enzyme is fully denatured. It is concluded, therefore, that charge, rather than steric restraint, is the determining factor for the differences seen in the modification patterns of the enzyme by these reagents. When enzyme was first labeled with 2,2'-dithiodipyridine and subsequently with p-mercuribenzoate, it was found that the latter, in a secondary process, will stoichiometrically react with the anion released by the former after the initial reaction with cysteine. The differences in reactivity of the cysteine residues toward the referred-to reagents have been exploited to specifically modify each of the three individual cysteine residues of pig muscle phosphoglucose isomerase.     

Human gastric lipase: a sulfhydryl enzyme

One sulfhydryl group was modified per mol of native human gastric lipase after incubation at pH 8.0 with 5,5'-dithiobis(2-nitrobenzoic acid) for 18 h or with 4,4'-dithiopyridine for 100 min. With both reagents a direct correlation was found between the modification of one sulfhydryl group and the loss of human gastric lipase activity. Incubation of human gastric lipase with a new hydrophobic sulfhydryl reagent dodecyldithio-5-(2-nitrobenzoic acid) in 30-fold molar excess, at pH 3.0, 5.0, and 8.0, induced immediate and complete human gastric lipase inactivation. Unlike 5,5'-dithiobis(2-nitrobenzoic acid) and 4,4'-dithiopyridine, dodecyldithio-5-(2-nitrobenzoic acid) almost instantaneously stopped the course of tributyrin hydrolysis by human gastric lipase. Human gastric lipase can thus be said to be a sulfhydryl enzyme.     

The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase

It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies.

1. Qualitative studies on the stability of rabbit erythrocyte purine nucleoside phosphorylase showed a marked decrease in the susceptibility of the enzyme to thermal inactivation and digestion by proteinases of different specificities in response to certain of its substrates. 2. The extent to which inosine stabilizes the enzyme against thermal and proteolytic inactivation is related in a quantitative manner to the concentration of this substrate; it is proposed that differences in the rates of inactivation of the enzyme may reflect substrate-induced conformational changes in the enzyme structure that could alter the binding properties of the enzyme in a kinetically significant way. 3. A synergistic effect in the stabilization of the enzyme is observed in response to both substrates, inosine and phosphate, when the enzyme is inactivated with Pronase. 4. In the presence of substrate an increased rate of inactivation after reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) is reported. 5. Differential-inactivation studies were also carried out with calf spleen purine nucleoside phosphorylase, and the results are discussed in relation to the kinetic properties displayed by this enzyme.     

Torpedo acetylcholinesterase is inactivated by thiol reagents

A number of sulphydryl reagents inhibit AChE of Torpedo california with pseudo-first-order kinetics, and inhibition can be retarded by quaternary ligands which bind at either the catalytic or peripheral anionic binding sites. Colorimetric determination with one of the inhibitory sulphydryl agents, 5,5'-dithiobis (2-nitrobenzoic acid), reveals the presence of a single thiol group per catalytic subunit; our data thus suggest that inhibition is achieved by reaction with the single free sulphydryl group of Cys231.     

Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding

1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.     

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the condensing component of chicken liver fatty acid synthase by iodoacetamide and 5,5''-dithiobis-(2-nitrobenzoic acid).

Chicken liver fatty acid synthase is inhibited by the thiol-modifying reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetamide. Total inactivation of the activity for fatty acid synthesis requires the modification of about 8 of the nearly 50 freely accessible thiol groups per molecule. The differential binding of iodo[14C]acetamide to phenylmethylsulphonyl fluoride-modified enzyme in the absence and in the presence of excess acetyl-CoA shows complete modification of one cysteine-SH site of the condensing enzyme and partial modification of the pantetheine-SH site for a total of approx. 1.4 mol of iodoacetamide bound per mol of enzyme. The reaction of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) generates disulphide cross-links for each molecule of the reagent added, but 95% of these cross-links are intrasubunit. Both the iodoacetamide- and 5,5'-dithiobis-(2-nitrobenzoic acid)-modified species catalyse all the component partial reactions of fatty acid synthesis except the condensation reaction. The results obtained with iodoacetamide show that in the dimeric fatty acid synthase modification of one cysteine-SH condensing site and/or one pantetheine-SH site per dimer is sufficient to affect inhibition of condensing activity and the activity for fatty acid synthesis, and are in accord with a recently proposed model for the mechanism of action of animal fatty acid synthases [Kumar (1982) J. Theor. Biol. 95, 263-283].     

Modification of creatine kinase by S-nitrosothiols: S-nitrosation vs. S-thiolation

Creatine kinase is reversibly inhibited by incubation with S-nitrosothiols. Loss of enzyme activity is associated with the depletion of 5,5'-dithiobis (2-nitrobenzoic acid)-accessible thiol groups, and is not due to nitric oxide release from RSNO. Full enzymatic activity and protein thiol content are restored by incubation of the S-nitrosothiol-modified protein with glutathione. S-nitroso-N-acetylpenicillamine, which contains a more sterically hindered S-nitroso group than S-nitrosoglutathione, predominantly modifies the protein thiol to an S-nitrosothiol via a transnitrosation reaction. In contrast, S-nitrosoglutathione modifies creatine kinase predominantly by S-thiolation. Both S-nitroso-N-acetylpenicillamine and S-nitrosoglutathione modify bovine serum albumin to an S-nitroso derivative. This indicates that S-thiolation and S-nitrosation are both relevant reactions for S-nitrosothiols, and the relative importance of these reactions in biological systems depends on both the environment of the protein thiol and on the chemical nature of the S-nitrosothiol.     

Assay of acyl-CoA:monoglyceride acyltransferase from rat small intestine using continuous recording spectrophotometry

Acyl-CoA:monoglyceride acyltransferase in microsomal preparations from the small intestine of the rat has been measured by means of continuous recording spectrophotometry. The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with CoA has been employed for this assay and optimal conditions for the reaction have been defined. One of the substrates, palmitoyl-CoA, inhibits the reaction even in modest concentrations. This inhibition is largely prevented by the addition of bovine serum albumin to the incubation medium. The reliability of the assay method was confirmed by comparison with the more cumbersome assay method that uses radioactive substrate.     

Effect of Ca2+ on conformation of actin in the F-actin--heavy meromyosin complex

The polarized fluorescence of intrinsic tryptophan residues and the birefringence of ghost muscle fibres of rabbit were measured during thin filaments binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains and to those devoid of them with a view of investigating conformational changes in F-actin. Ca2+ binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains was shown to affect the character of these changes during the formation of the F-actin - heavy meromyosin complex.     

High concentrations of aldehydes slow the reaction of cytoplasmic aldehyde dehydrogenase with thiol-group modifiers.

High concentrations of aldehydes slow the inactivation of cytoplasmic aldehyde dehydrogenase by disulfiram and also slow the reaction of the enzyme with 2,2'-dithiodipyridine. It is concluded that a low-affinity aldehyde-binding site is probably the site at which thiol-group modifiers react with aldehyde dehydrogenase, as well as being the active site for hydrolysis of 4-nitrophenyl acetate.