A novel enhancing mechanism for hydrogen sulfide-producing activity of cystathionine beta-synthase

H2S is produced from cysteine by cystathionine beta-synthase (CBS) in the brain and functions as a neuromodulator. Although the production of H2S is regulated by Ca2+ and calmodulin in response to neuronal excitation, little is known about the molecular mechanism for the regulation in CBS activity. Here we show that four cysteine residues of CBS are involved in the regulation of its activity in the presence of Ca2+ and calmodulin. Sodium nitroprusside (SNP), a modifying agent for cysteine residues, enhances CBS activity, whereas N-ethylmaleimide, an alkylating agent for cysteine residues, completely abolished the effect of SNP. Site-directed mutagenesis of the 13 cysteine residues of CBS identified four cysteine residues that are involved in the regulation of CBS activity by SNP, and two of the four residues are involved in the regulation of the basal CBS activity. The enhancement of CBS activity by SNP is independent of nitric oxide production. In the presence of Staphylococcus aureus alpha-hemolysin, which permeabilizes the cell membrane, exogenously applied SNP enhances the activity of CBS in intact cells. The present study demonstrates a novel mechanism for the regulation of CBS activity and provides a possible therapeutic application of SNP for the diseases in which CBS activity is deficient.     

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.     

Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification

The integral V(0) domain of the vacuolar (H(+))-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V(0) that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.     

Peptide-Formation on Cysteine-Containing Peptide Scaffolds

Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Gly_n-Cys-Glu₁₀, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25–65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu₁₀ in the presence of Arg₅ leads to the formation of Cys-Arg₅ in 50% yield. The significance of these results for prebiotic chemistry is discussed.     

Affinity labelling of yeast and liver alcohol dehydrogenases with the NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine.

The NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo first order. The stoichiometry is one male inactivator incorporated per mole enzyme subunit. The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labelling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labelled on the essential cysteine residue 43.     

Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase

The specific chemical modification by sodium cyanate of highly reactive cysteine residues at pH 7.5 in pig kidney fructose 1,6-bisphosphatase results in the reversible loss of activation of the enzyme by monovalent cations. No loss of activation by potassium ions occurs when modification is carried out in the presence of fructose 2,6-bisphosphate. The effect of Mg2+ on native and cyanate-modified enzyme activities implicates the above cysteine residue as being directly linked to the inhibition by both the divalent cation and fructose 2,6-bisphosphate. Incorporation of [14C]cyanate to the enzyme shows that the blockage of two reactive residues per tetramer is sufficient to eliminate the activation of the enzyme by K+.     

α-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide

alpha-Crystallin was carboxymethylated with radioactive iodoacetic acid in the presence of 7.6m-urea and then separated into six major fractions by chromatography on DEAE-cellulose in 7m-urea. Based on the amino acid compositions, specific radioactivities and sodium dodecyl sulphate-gel electrophoresis of the fractions, it was concluded that alpha-crystallin contains at least four different subunits: DU1A and DU1B, containing no cysteine; a third component represented by DU2B and DU3 containing one cysteine one cysteine residue per subunit; and DU4, which probably contains two residues of cysteine per subunit. Subunit DU1A was shown to be of sufficient purity for sequence studies. Cyanogen bromide cleavage yielded two peptides, CB-1 and CB-2, in approximately equal amounts as expected. The sum of the molecular weights and amino acid compositions of the peptides were both in excellent agreement with the results obtained for subunit DU1A. The amino acid sequence of the first sixteen residues of peptide CB-1 is: Ser-Leu-Thr-Lys-Asp-Phe-Asp-Glu-Val-Asn-Ile-Asp-Val-Ser-His-Phe-. The sequence of the first seventeen residues of peptide CB-2 is: Asp-Ile-Ala-Ile-Ser-His-Pro-Trp-Ile-Arg-Pro-Ser-Phe-Phe-Glu-Phe-His-. The N-terminal sequence of subunit DU1A was shown to be N-acetylmethionine followed by peptide CB-2.     

Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl)maleimide

N-(7-Dimethylamino-4-methylcoumarynyl)maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli. Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J.S., et al. (1985) Nucl. Acids Res. 13, 2063-2074). When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified. This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e., the thiolate) within the active site. Although this is likely to be an important chemical attribute of many thiol-based enzymes, including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pK(a) values have been determined experimentally. Presented here is a new technique for determining the pK(a) value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d(5)) and protiated (d(0)) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pK(a) values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and greater than 10.0, in good agreement with previous reports using chemical modification approaches. This technique allows the pK(a) of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest.     

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.     

The role of cysteine residues in the oxidation of ferritin

We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999-1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates.     

Conformation and microenvironment of the active site of a low molecular weight 1,4-beta-D-glucan glucanohydrolase from an alkalothermophilic Thermomonospora sp.: involvement of lysine and cysteine residues

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.     

Modification of plasma membrane protein cysteine residues by ADP-ribose in vivo

Proteins can be post-translationally modified by ADP-ribose. Previously, two classes of ADP-ribosyl protein linkages have been detected in vivo which have chemical properties indistinguishable from ADP-ribosyl arginine and ADP-ribosyl glutamate or aspartate. Reported here is the detection of a third class of endogenous ADP-ribosyl protein linkage. This class is chemically indistinguishable from ADP-ribose linked to cysteine residues by a thioglycosidic bond. The distribution of ADP-ribosyl cysteine residues was studied in subcellular fractions of rat liver. Proteins modified on cysteine were detected only in the plasma membrane fraction. Pertussis toxin is known to disrupt signal transduction of ADP-ribosylation of cysteine residues of plasma membrane GTP binding proteins. The results described here raise the interesting possibility that the endogenous modification of plasma membrane protein cysteine residues may be involved in signal transduction.     

Single amino acid substitutions at conserved residues of human interferon-alpha can effect antiviral specific activity

Site-specific in vitro mutagenesis was used to direct various amino acid substitutions at conserved positions within the sequence of human interferon-alpha 1 (IFN-alpha 1). The antiviral specific activity of IFN-alpha 1, expressed in M13 as a fusion protein [IFN-alpha 1 (phi WT)], could be altered by single amino acid substitutions. The substitution of glycine for tyrosine at position 123 results in a loss of more than 99% of the antiviral specific activity on human cells, but causes no significant change in the antiviral specific activity on primary bovine cells. The tyrosine at position 123 is thus implicated in determining human cell specificity. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two dulsulphide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. IFN-alpha 1 (phi WT) carrying three serine for cysteine substitutions at positions 1, 86 and 99 retains 23% of the antiviral specific activity of IFN-alpha 1 (phi WT) on human cells. However, the antiviral activity on bovine cells is not significantly affected by this modification. The presence of the disulphide bridge between residues 1 and 99 thus appears to be required for full antiviral activity on human but not bovine cells. A single serine for cysteine substitution at position 29 reduces the antiviral specific activity on both human and bovine cells by some 95%. This data shows that the disulphide bridge between residues 29 and 139 is critical for the antiviral activity of IFN-alpha's.     

The occurrence of disulphide bonds in purified clathrin light chains.

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Mutational analysis of disulfide bridges in the Mr 46,000 mannose 6-phosphate receptor. Localization and role for ligand binding

Formation of intramolecular disulfide bonds is a key step in the early maturation of newly synthesized Mr 46,000 mannose 6-phosphate receptors to acquire ligand-binding activity (Hille, A., Waheed, A., and von Figura, K. (1990) J. Cell Biol. 110, 963-972). The luminal domain of the receptor, which carries the ligand-binding site, contains 6 cysteine residues. We have analyzed the function of individual cysteine residues for the ligand-binding conformation by exchanging cysteine for glycine. In each case, the replacement of cysteine resulted in a complete loss of binding activity, indicating that all 6 luminal cysteine residues are required for the ligand-binding conformation. The cysteine mutants displayed a greatly reduced immunoreactivity, decreased stability, and a blocked or delayed transport to the trans Golgi. The glycosylation pattern allowed the distinguishing of three phenotypes, each of which was represented by one pair of cysteine mutants. Based on the assumption that replacement of either of the 2 cysteine residues forming a disulfide bond results in an identical phenotype, we postulate that disulfide bonds are formed between Cys-32 and Cys-78 and between Cys-132 and Cys-167, as well as between Cys-145 and Cys-179. This assumption was supported by the observation that the simultaneous exchange of the 2 cysteine residues of a putative pair resulted in the same phenotypes as the single exchange of either of the 2 cysteine residues.