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PubMed1999

The FMN to heme electron transfer in cytochrome P450BM-3. Effect of chemical modification of cysteines engineered at the FMN-heme domain interaction site.

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro(382)-Gln(387) peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys(372) and Cys(387) resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys(104) caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.

Bacillus megaterium↗
PubMed1992

Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel.

It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free -SH group of proteins, forming a cysteinyl-S-propionamide adduct. When beta-lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu-149 to Ile-162. Residue No. 160 was found to be a cysteinyl-S-propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including -NH2 terminus and free -NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the -SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.

Acrylamide↗
PubMed2001

Mutagenesis and derivatization of human vesicle monoamine transporter 2 (VMAT2) cysteines identifies transporter domains involved in tetrabenazine binding and substrate transport.

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. Photoaffinity labeling, chimera analysis, and mutagenesis have identified functionally important amino acids and provided some information regarding structure and ligand binding sites. To extend these studies, we engineered functional human VMAT2 constructs with reduced numbers of cysteines. Subsets of cysteines were discovered, which restore function to an inactive cysteine-less human VMAT2. Replacement of three transmembrane (TM) cysteines together (net removal/replacement of three atoms) significantly enhanced monoamine transport. Cysteine modification studies involving single and combination cysteine mutants with methanethiosulfonate ethylamine revealed that [(3)H]dihydrotetrabenazine binding is > 90% inhibited by modification of two sets of cysteines. The primary target (responsible for approximately 80% of inhibition) is Cys(439) in TM 11. The secondary target (responsible for approximately 20% of inhibition) is one or more of the four non-TM cysteines. [(3)H]Dihydrotetrabenazine protects against modification of Cys(439) by a 10,000-fold molar excess of methanethiosulfonate ethylamine, demonstrating that Cys(439) is either at the tetrabenazine binding site, or conformationally linked to tetrabenazine binding. Supporting a direct effect, the position of tetrabenazine-protectable Cys 439 is consistent with previous mutagenesis, chimera, and photoaffinity labeling data, demonstrating involvement of TM 10-12 in a tetrabenazine binding domain.

Animals↗
PubMed1994

Modification of cysteine residues with N-methyl iodoacetamide.

Cysteine residues derivatized with N-methyl iodoacetamide (MIAA) can be analyzed by the Edman sequencing with a high degree of reliability. By HPLC, the phenylthiohydantoin (PTH) derivative of MIAA-modified cysteine eluted between dimethylphenylthiourea and PTH-Ala--a wide gap which is not occupied or interfered with by any other by-products or PTH amino acids. During extended Edman degradation, the recovery of PTH derivative of MIAA-modified Cys was as quantitative and reproducible as that of other stable PTH derivatives such as Ala, Val, and Leu.

Amino Acids↗
PubMedDate not supplied

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function.

Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.

Acyl Coenzyme A↗
PubMed1998

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.

Acrylamide↗
PubMed1984

Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid.

Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.

Alcohol Dehydrogenase↗
PubMed1989

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893.

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with diazonium-1H-tetrazole.

Diazonium-1H-tetrazole was tested as a potential active-site-directed reagent for amino acid residues involved in catalysis by alcohol dehydrogenase. In a novel reaction with a protein, diazonium-1H-tetrazole inactivated the enzyme selectively, and almost stoichiometrically, but reacting with the sulfur of a cysteine residue, Cys-174. As a model compound, the tetrazole adduct of free cysteine was prepared. Elementary and spectral analyses of the adduct were consistent with the structure 5-tetrazoleazo-S-cysteine. The adduct absorbs light with a maximun at 316 nm, and is destroyed by irradiation at this wavelength. The inactivated enzyme still bound NADH as determined by difference spectroscopy, but did not enhance the fluorescence of the bound NADH as did native enzyme. X-ray crystallographic studies of free enzyme have shown that Cys-174 coordinates the zinc at the active site (Eklund, H., Nordström, B., Zeppezauer, E., Söderlund, G., Ohlsson, I., Boiwe, T., and Brändén, C-I. (1974), FEBS Lett. 44, 200-204). The modified enzyme is probably inactive because the large, negatively charged tetrazole ring interferes sterically or electrostatically with the binding of substrates or with hydride transfer.

Alcohol Oxidoreductases↗
PubMed1996

Activation of Sulfolobus solfataricus alcohol dehydrogenase by modification of cysteine residue 38 with iodoacetic acid.

Reaction of thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus with iodoacetate at pH 9.0 and 37 degrees C significantly increases the oxidation rate of aliphatic and aromatic alcohols and decreases the reduction rate of aromatic aldehydes. The archaeal ADH is chemically modified and activated in a Michaelis-Menten-type reaction, where one molecule of the reagent binds per active site. NAD+ in micromolar concentration protects the enzyme against the inhibitor in an uncompetitive manner, while imidazole significantly increases the extent of the activation. Carboxymethylation selectively modifies one out of five cysteine residues per subunit, namely, Cys 38, located in the catalytic site, as determined by peptide and sequence analysis, and enhances by up to 25-fold the oxidation rate of benzyl alcohol. Carboxymethylated SsADH is less thermostable and shows a temperature optimum 30 degrees C lower than that of the native enzyme. The carboxymethylated enzyme exhibits a lower affinity toward the oxidized and reduced coenzyme. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 are 60- and 200-fold higher, respectively, compared to the native enzyme. The significant isotope effect in alcohol oxidation suggests that hydride transfer partially limits the turnover rate of the reaction catalyzed by the modified enzyme, whereas the rate-limiting step for the native enzyme is NADH dissociation. Carboxymethylated enzyme probably gives higher maximum velocities of oxidation because of the faster dissociation of the modified enzyme-coenzyme complex.

Alcohol Dehydrogenase↗
PubMed2003

Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to alpha-carbons of phenylalanine and threonine.

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]phenylalanine and [U-(13)C,(15)N]threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. Such posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is very unusual in natural peptides or proteins. Subtilosin A (1) belongs to a new class of bacteriocins.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.

Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.

Amino Acid Sequence↗
PubMed2000

Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica.

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.

4-Chloromercuribenzenesulfonate↗
PubMed2005

Regulation of constitutive neutrophil apoptosis by the alpha,beta-unsaturated aldehydes acrolein and 4-hydroxynonenal.

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.

Acrolein↗