Photolabeling of CheR methyltransferase with S-adenosyl-L-methionine (AdoMet). Studies on the AdoMet binding site.

CheR methyltransferase from Salmonella typhimurium was directly photolabeled with S-adenosyl-L-[methyl-3H]methionine. The labeled protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was detected by fluorography. The methylase-S-adenosyl-L-methionine adduct was found to be stable under the experimental conditions employed. Labeling was found to be a function of the concentration of enzyme, S-adenosyl-L-methionine (AdoMet), and the intensity and time of UV irradiation. The extent of labeling and protein methylation was found to be inhibited by S-adenosyl-L-homocysteine, S-adenosyl-L-ethionine, and sinefungin, which are known to compete with AdoMet for the same binding site on the enzyme. Our earlier data showed that the enzyme has 2 cysteine residues and that these are important for enzyme activity. Here, we show that sulfhydryl reagents inhibit the photolabeling of the substrate to the enzyme, indicating the presence of cysteine in the vicinity of the substrate-binding site. We also found that when Cys31 was modified to Ser, no photolabeling of CheR was observed, whereas a modification of Cys229 to Ser had little effect on the ability of AdoMet to label the enzyme. This suggests that Cys31 is located at or near AdoMet-binding site. The labeled protein was cleaved at tryptophan residues, generating two major fragments, each containing 1 cysteine residue. SDS-PAGE and fluorography of the cleaved products indicated the presence of the label being associated with the Cys31 fragment. Similar results were obtained when the labeled protein was cleaved at glutamic acid residues using V8 protease. A tryptic digest of the labeled protein showed two radioactive peptide peaks when subjected to separation on reverse phase high pressure liquid chromatography. The labeled peptides were further digested to free amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that Cys31 may be involved with substrate binding, as well as with catalysis.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.     

Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis.

The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239. Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents. The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification. Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair. Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine. The mutant proteins were overexpressed and purified, and their kinetics were studied. The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity. The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair. However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.     

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

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

Structural and functional roles of cysteine residues of Bacillus polymyxa beta-amylase.

Bacillus polymyxa beta-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91 V enzymes followed by amino acid sequencing. Therefore, only Cys323 contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme. C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.

The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.     

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.     

Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis

Cholesterol 7alpha-hydroxylase (cholesterol-NADPH oxidoreductase, EC 1.14.13.17, 7alpha-hydroxylating) is known to have extremely sensitive sulfhydryl group(s). It is believed that a cysteine residue that has a sulfhydryl group plays an important role in the decrease of this enzyme activity. The amino acid sequences of cholesterol 7alpha-hydroxylase of five different mammalian species, human, rat, rabbit, hamster and mouse, revealed that these mammalian species contain eight cysteine residues that are well conserved. To identify which cysteine residues are responsible for the extremely high lability, we used the technique of the site-directed mutagenesis. Eight mutated genes of human cholesterol 7alpha-hydroxylase in which one codon for a cysteine residue was changed to that for alanine were prepared and expressed in COS-1 cells. The protein mass and enzyme activity of cholesterol 7alpha-hydroxylse obtained from these eight mutated genes were determined. While all mutated genes expressed the enzyme mass, two mutated genes did not express protein capable of catalyzing 7alpha-hydroxylation of cholesterol: in one mutant a codon for the 7th cysteine residue (Cys 444) was substituted to that for alanine and in the other mutant a codon for the 8th cysteine residue (Cys 476) was changed similarly. These results suggest that the 7th and 8th cysteine residues are important for expression of the enzyme activity. Based on the fact that Cys 444 exists in the heme binding region, Cys 476 was suggested to be responsible for enzyme lability.     

Active site of the enzyme which demethylates receptors during bacterial chemotaxis

The CheB methylesterase catalyzes the hydrolysis of glutamyl methyl esters in bacterial chemoreceptor proteins. Studies with residue-specific inhibitors suggest that a cysteine residue is required. The nucleotide sequence of the cheB gene predicts a 349-amino acid protein with cysteine residues at positions 207 and 309. Oligonucleotide-directed mutagenesis was used to change each cysteine to an alanine. Whereas the Cys207-Ala mutation had essentially no effect on esterase activity, the Cys309-Ala mutation caused a complete inactivation of the enzyme. Cys309 is located adjacent to a sequence of amino acids which is characteristic of the beta-alpha-beta motif found in a number of nucleotide binding proteins associated with receptor function in vertebrate tissues. A central feature of this structure is Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this region (Gly284) to a valine also caused a complete loss of esterase activity.     

Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase.

Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V(max) and K(m) values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained approximately 66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.     

Reactive cysteine in the active-site motif of Bacteroides thetaiotaomicron dipeptidyl peptidase III is a regulatory residue for enzyme activity

Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.     

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.     

Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide.

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and B?ck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.     

Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase

The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.     

The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

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.