Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human gamma-glutamyl hydrolase.

ABSTRACT: Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

SUBMITTER: Chave KJ

PROVIDER: S-EPMC1220585 | biostudies-other | 1999 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

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

Effects of Site-Directed Mutagenesis of Cysteine on the Structure of Sip Proteins.

Project description:Bacillus thuringiensis, a gram-positive bacteria, has three insecticidal proteins: Vip (vegetative insecticidal protein), Cry (crystal), and Sip (secreted insecticidal protein). Of the three, Sip proteins have insecticidal activity against larvae of Coleoptera. However, the Sip1Aa protein has little solubility in the supernatant because of inclusion bodies. This makes it more difficult to study, and thus research on Sip proteins is limited, which hinders the study of their mechanistic functions and insecticidal mechanisms. This highlights the importance of further investigation of the Sip1Aa protein. Disulfide bonds play an important role in the stability and function of proteins. Here, we successfully constructed mutant proteins with high insecticidal activity. The tertiary structure of the Sip1Aa protein was analyzed with homologous modeling and bioinformatics to predict the conserved domain of the protein. Cysteine was used to replace amino acids via site-directed mutagenesis. We successfully constructed Sip149-251, Sip153-248, Sip158-243, and Sip178-314 mutant proteins with higher solubility than Sip1Aa. Sip153-248 and Sip158-243 were the most stable compared to Sip1Aa, followed by Sip149-251 and Sip178-314. The insecticidal activity of Sip153-248 (Sip158-243) was 2.76 (2.26) times higher than that of Sip1Aa. The insecticidal activity of Sip149-251 and Sip178-314 did not differ significantly from that of Sip1Aa. Basic structural properties, physicochemical properties, and the spatial structure of the mutation site of Sip1Aa and the mutant proteins were analyzed. These results provide a molecular basis for using Sip1Aa to control Coleopteran insects and contribute to the study of the Sip1Aa insecticidal mechanism.

| S-EPMC9100928 | biostudies-literature

Gamma-glutamyl hydrolase: kinetic characterization of isopeptide hydrolysis using fluorogenic substrates.

Project description:Gamma-glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-gamma-glutamate derivatives of folate cofactors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-gamma-glutamate and (4,4-difluoro)glutamyl-gamma-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a approximately 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is approximately 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under nonsaturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction.

| S-EPMC2562270 | biostudies-literature

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

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

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

Newly Identified Essential Amino Acids Affecting Chlorella ellipsoidea DGAT1 Function Revealed by Site-Directed Mutagenesis.

Project description:Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in the synthesis of triacylglycerol (TAG), the most important form of energy storage in plants. Some residues have previously been proven to be crucial for DGAT1 activity. In this study, we used site-directed mutagenesis of the CeDGAT1 gene from Chlorella ellipsoidea to alter 16 amino acids to investigate effects on DGAT1 function. Of the 16 residues (L482R, E542R, Y553A, G577R, R579D, Y582R, R596D, H603D, H609D, A624R, F629R, S632A, W650R, A651R, Q658H, and P660R), we newly identified 5 (L482, R579, H603, A651, and P660) as being essential for DGAT1 function and 7 (E542, G577, R596, H609, A624, S632, and Q658) that significantly affect DGAT1 function to different degrees, as revealed by heterologous expression of the mutants in yeast strain INVSc1. Importantly, compared with CeDGAT1, expression of the mutant CeDGAT1Y553A significantly increased the total fatty acid and TAG contents of INVSc1. Comparison among CeDGAT1Y553A, GmDGAT1Y341A, AtDGAT1Y364A, BnDGAT1Y347A, and BoDGAT1Y352A, in which tyrosine at the position corresponding to the 553rd residue in CeDGAT1 is changed into alanine, indicated that the impact of changing Y to A at position 553 is specific for CeDGAT1. Overall, the results provide novel insight into the structure and function of DGAT1, as well as a mutant gene with high potential for lipid improvement in microalgae and plants.

| S-EPMC6274981 | biostudies-literature

Epigenetic regulation of human gamma-glutamyl hydrolase activity in acute lymphoblastic leukemia cells.

Project description:Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.

| S-EPMC1559484 | biostudies-literature

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.

Project description:Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.

| S-EPMC2175162 | biostudies-literature

Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis.

Project description:The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331----Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331----Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

| S-EPMC1151543 | biostudies-other

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