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1.
Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis 总被引:2,自引:0,他引:2
H Kato T Tanaka T Nishioka A Kimura J Oda 《The Journal of biological chemistry》1988,263(24):11646-11651
Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents. 相似文献
2.
Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant. 相似文献
3.
Mohamed Ali Borgi Moez Rhimi Nushin Aghajari Mamdouh Ben Ali Michel Juy Richard Haser Samir Bejar 《Biologia》2009,64(5):845-851
The implication of the original alanine 63 (Ala63) and the unique cysteine 306 (Cys306) residues in the thermostability of
the Streptomyces sp. SK glucose isomerase (SKGI) were investigated by site-directed mutagenesis and homology modelling. The Cys306 to Ala
mutation within SKGI dramatically affected its thermal stability by decreasing the half-life from 80 to 15 min at 90°C while
the Ala63 to Ser replacement shifted this half-life to 65 min. The electrophoretic analysis proves that the residue Cys306
participates in oligomerization of the SKGI. Its stabilizing role is materialized by hydrogen bonds established with arginines
at positions 284 and 259, as deduced from the constructed three-dimensional model. We have also shown that the presence of
an Ala63 instead of Ser63 seems to be more suitable for enzyme thermostability by maintaining hydrophobic pocket that contributes
to the protection of the enzyme active site. 相似文献
4.
5.
Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines 总被引:1,自引:0,他引:1
R A Hallewell K C Imlay P Lee N M Fong C Gallegos E D Getzoff J A Tainer D E Cabelli P Tekamp-Olson G T Mullenbach 《Biochemical and biophysical research communications》1991,181(1):474-480
Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability. 相似文献
6.
We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na(+)/H(+) exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N-tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N-tail. This approach may provide evidence for the interdomain interaction in NHE1. 相似文献
7.
Ito G Ariga H Nakagawa Y Iwatsubo T 《Biochemical and biophysical research communications》2006,339(2):667-672
A significant proportion of early onset parkinsonism is inherited as an autosomal-recessive trait (AR-EP). DJ-1 was identified as one of the causative genes for AR-EP (PARK7), and DJ-1 protein has been implicated in oxidative stress response through oxidation of one of the three cysteine residues (i.e., Cys106). However, the individual roles of these cysteine residues remained unclear. We show by a systematic mutagenesis analysis that Cys46 and Cys53 of DJ-1, but not Cys106, are susceptible to S-nitrosylation in vitro as well as in cultured cells. Furthermore, alanine substitution of Cys46 diminished dimerization of DJ-1, a fundamental feature of this protein. These results indicate that distinct cysteine residues of DJ-1 harbor differential roles in relation to its structure and function. 相似文献
8.
Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry 总被引:1,自引:0,他引:1
Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate. 相似文献
9.
Neurokinin-2 receptor (NK(2)R) binding of [(3)H]-SR48968, a piperidinyl antagonist, is inhibited by methanethiosulfonate ethylammonium (MTSEA) in a time- and concentration-dependent manner. By the systematic alanine replacement of putative loop and transmembrane region cysteine residues (Cys(4), Cys(81), Cys(167), Cys(262), Cys(281), Cys(308), and Cys(309)), we have determined that MTSEA perturbs [(3)H]-SR48968 binding by modifying Cys(167) in transmembrane helix 4. Data were substantiated using glycine, serine, and threonine substitutions of Cys(167). MTSEA preferentially modifies cysteine residues that are in proximity to a negatively charged environment. Hence, aspartate and glutamate residues were systematically substituted with leucine or valine, respectively, and the inhibitory effects of MTSEA on [(3)H]-SR48968 binding were reevaluated to determine those acidic residues close to the MTSEA binding crevice. Most significantly, substitution of Asp(5) in the receptor's extreme N-terminus abolished the effects of MTSEA on [(3)H]-SR48968 binding. Therefore, our data would suggest close association of the extreme N-terminus with the extracellular surfaces of helices 4 and 3 in the NK(2)R in forming a binding crevice for MTSEA. The inhibition of SR48968 binding appears to result from loss of the SR48968 binding conformation of Gln(166) induced by MTSEA when it is coupled to Cys(167). Hence, it is proposed that there is mutually exclusive hydrogen bonding of SR48968 and MTSEA to Gln(166). 相似文献
10.
Velasco-García R Zaldívar-Machorro VJ Mújica-Jiménez C González-Segura L Muñoz-Clares RA 《Biochemical and biophysical research communications》2006,341(2):408-415
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa. 相似文献
11.
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. 相似文献
12.
Role of cysteine residues in structural stability and function of a transmembrane helix bundle 总被引:1,自引:0,他引:1
Karim CB Paterlini MG Reddy LG Hunter GW Barany G Thomas DD 《The Journal of biological chemistry》2001,276(42):38814-38819
To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB. 相似文献
13.
Li S Kimura M Takashima T Hayashi K Inoue K Ishiguro R Sugisaki H Maruyama K 《Bioscience, biotechnology, and biochemistry》2007,71(2):449-457
4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent. 相似文献
14.
S M Miller V Massey D Ballou C H Williams M D Distefano M J Moore C T Walsh 《Biochemistry》1990,29(11):2831-2841
A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes. 相似文献
15.
Kim BM Schultz LW Raines RT 《Protein science : a publication of the Protein Society》1999,8(2):430-434
Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein. 相似文献
16.
Sulfide:quinone oxidoreductase from the acidophilic and chemolithotrophic bacterium Acidithiobacillus ferrooxidans was expressed in Escherichia coli and crystallized, and its X-ray molecular structure was determined to 2.3 Å resolution for native unbound protein in space group P42212 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.3 Å and 2.05 Å resolutions, respectively, in space group P6222 . The enzymatic reaction catalyzed by sulfide:quinone oxidoreductase includes the oxidation of sulfide compounds H2S, HS−, and S2− to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; these oxidations are coupled to the reduction of ubiquinone or menaquinone. The enzyme comprises two tandem Rossmann fold domains and a flexible C-terminal domain encompassing two amphipathic helices that are thought to provide for membrane anchoring. The second amphipathic helix unwinds and changes its orientation in the hexagonal crystal form. The protein forms a dimer that could be inserted into the membrane to a depth of approximately 20 Å. It has an endogenous flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the N-terminal domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues situated on the re side of the FAD cofactor form a branched polysulfide bridge. Cys356 disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The third essential cysteine Cys128 is not modified in these structures; its role is likely confined to the release of the polysulfur product. 相似文献
17.
Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria. The enzyme is an alpha 2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 10(4)-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains. 相似文献
18.
Saeki T Munetaka Y Ueda K Iwami K Kanamoto R 《Bioscience, biotechnology, and biochemistry》2007,71(8):1865-1872
Although ileal and hepatic Na(+)-dependent bile acid transporters (SLC10A2 and SLC10A1 respectively) share structural similarities, the mutation of conserved amino acids often has distinct effects on them. We have identified two Cys residues in mouse Slc10a2 (Cys(51) and Cys(106)) the replacement of which by Ala remarkably reduces taurocholic acid (TCA) transport. Although Cys(51) is conserved in Slc10a1 as Cys(44), Ala substitution gave no apparent difference in TCA uptake. Here, we further analyzed the kinetics of TCA uptake and cell surface localization of these mutants. The C51A and C106A mutants of Slc10a2 showed significantly reduced TCA uptake, while no apparent difference in TCA uptake was observed for the Slc10a1-C44A mutant. The K(m) values for TCA uptake by these mutants were comparable, suggesting that these residues are not involved in the interaction with TCA. 相似文献
19.
The formation of a complex between the specific chaperone-type protein HypC and the precursor form of the large subunit HycE in the maturation pathway of hydrogenase 3 from Escherichia coli has been studied by targeted replacement of amino acids in both proteins. HypC and its homologs contain the motif MC(L/I/V)(G/A)(L/I/V)P at the amino terminus, from which the methionine residue is post-translationally removed. The exchange of the cysteine residue led to complete loss of the ability to interact with the precursor form of HycE, but replacement of the proline residue had no effect. Site-directed replacement of the conserved cysteine residues in HycE involved in nickel binding was also performed. Exchange of Cys(241) resulted in the inability of the HycE variant to interact with HypC and to incorporate nickel. The variants of HycE in which Cys(244) and Cys(531) were replaced by alanine residues were unable to incorporate nickel, although the mutated proteins could interact with HypC. Intriguingly, the precursor of HycE in which the Cys(534) residue was exchanged could form the complex with HypC, could incorporate nickel, and was C-terminally processed, but it delivered an inactive enzyme. Our findings are in favor of a model in which binding of HypC masks Cys(241); Cys(244) and Cys(531) bind the iron and nickel moieties, respectively; and C534 closes the bridge between the two metals after C-terminal processing has taken place. 相似文献
20.
Xu Y Kakhniashvili DA Gremse DA Wood DO Mayor JA Walters DE Kaplan RS 《The Journal of biological chemistry》2000,275(10):7117-7124
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. 相似文献