Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase. Identification of a cysteine residue critical for the oligomeric state.

ABSTRACT: We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns. The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting. No monomeric forms of the enzyme were detected in any case. Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase. Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms.

SUBMITTER: Mingorance J

PROVIDER: S-EPMC1217272 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Identification of an important cysteine residue in human glutamate-cysteine ligase catalytic subunit by site-directed mutagenesis.

Project description:Glutamate-cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110+/-12 micromol/h per mg of protein compared with 370+/-20 micromol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.

| S-EPMC1219919 | biostudies-other

Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue.

Project description:cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3' end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.

| S-EPMC1134902 | biostudies-other

Site directed mutagenesis of Schizosaccharomyces pombe glutathione synthetase produces an enzyme with homoglutathione synthetase activity.

Project description:Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5? and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k(m) 0,19; wild-type) to ?-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for ?-Ala (k(m) 0,07) and lowered the affinity for Gly (k(m) 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k(m) 0,23) as a substrate, while affinity to Gly was preserved (k(m) 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.

| S-EPMC3473041 | biostudies-literature

Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase.

Project description:N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.

| S-EPMC3820426 | biostudies-literature

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.

Project description:Redox active cysteine residues including ?Cys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of ?Cys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active ?Cys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of ?Cys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at ?19 on the exposed surface of the ?-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

| S-EPMC6126208 | biostudies-literature

Identification of an active site cysteine residue in human type I Ins(1,4,5)P3 5-phosphatase by chemical modification and site-directed mutagenesis.

Project description:Chemical modification using thiol-directed agents and site-directed mutagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human type I Ins(1,4,5)P3 5-phosphatase. Irreversible inhibition of enzymic activity is provoked by chemical modification of the enzyme by N-ethylmaleimide (NEM), 5,5'-dithio-2-nitrobenzoic acid, iodoacetate and to a much smaller extent by iodoacetämide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0.9 mol of NEM per mol of enzyme. A single [14C]NEM-modified peptide was isolated after alpha-chymotrypsin proteolysis of the radiolabelled enzyme and reverse-phase HPLC. Sequence analysis of the active site-labelled peptide (i.e. MNTRCPAWCD) demonstrated that Cys348 contained the radiolabel. Furthermore two mutant enzymes were obtained by site-directed mutagenesis of the cysteine residue to serine and alanine respectively. Both mutant enzymes had identical UV CD spectra. The two mutants (i.e. Cys348-->Ser and Cys348-->Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part of the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P3 5-phosphatase. This cysteine residue is part of a sequence 10 amino acids long that is well conserved among the primary structures of inositol and phosphatidylinositol polyphosphate 5-phosphatases.

| S-EPMC1217914 | biostudies-other

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis.

Project description:Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.

| S-EPMC2517115 | biostudies-literature

Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis.

Project description:In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabilities at a wide range of temperatures. The Km values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine were 15.7?±?1.1, (25.2?±?1.5)?×?10-5 and 32.6?±?1.7?mM, and the kcat were 17.0?±?0.6, 9.14?±?0.12 and 30.5?±?1.0?s-1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efficiency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50?°C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its flexibility due to the breaking of hydrogen bonds between product and enzyme. This also facilitated the enzyme to increase its cold adaptability as indicated by higher residual activity shown at 0?°C. Thus, replacement of Ala to Ser54 played a pivotal role to enhance the activities and stabilities at a wide range of temperatures.

| S-EPMC6199252 | biostudies-literature

Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308.

Project description:Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Douglas, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-435]. This residue was replaced with serine and alanine residues by site-directed mutagenesis. The mutated genes expressed proteins with low but finite ICL activity, which co-migrated with wild-type ICL on both SDS/ and native PAGE. The mutant proteins were purified and characterized. Fluorimetry and c.d. in both the near- and the far-u.v. regions showed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C195A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-fold increases in the Km for isocitrate, while their kcat. values showed 30- and 100-fold decreases respectively. The effect of pH on the kinetic properties of the wild-type and mutant ICLs was investigated. The results showed that the response of the mutant enzymes to pH was simpler than that of the wild-type. For the mutants, ionisation of a group with a pKa of approx. 7.8 affected the Km for isocitrate and kcat.. For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to by cysteine-195. The results are consistent with the view that the previously identified group with a pKa of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.

| S-EPMC1136455 | biostudies-other

Construction and functional analyses of a comprehensive sigma54 site-directed mutant library using alanine-cysteine mutagenesis.

Project description:The sigma(54) factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. sigma(54) is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define sigma(54) residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of sigma(54) was constructed by alanine-cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)sigma(54) were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of sigma(54) were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased sigma(54) isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after sigma(54) isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the sigma(54)-core RNAP interface changes upon activation.

| S-EPMC2715252 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data