Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue.
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ABSTRACT:
SUBMITTER: Humm A
PROVIDER: S-EPMC1218254 | biostudies-other | 1997 Mar
REPOSITORIES: biostudies-other
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Project description:Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.
| S-EPMC1138844 | biostudies-other
Project description:GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.
| S-EPMC1134771 | biostudies-literature
Project description:Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.
| S-EPMC1221021 | biostudies-other
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
Project description:The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5'-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.
| S-EPMC1137939 | biostudies-other
Project description:Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational ?-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The ?-lysyl EF-P modification pathway is conserved among only 26-28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl-EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli ?-lysyl-EF-P. The possible reasons for the unique requirement of rhamnosyl-EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others.
| S-EPMC4739656 | biostudies-literature
Project description:BackgroundThiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.ResultsBased on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.ConclusionsBased on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.
| S-EPMC4215014 | biostudies-literature
Project description:Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.
| S-EPMC1221134 | biostudies-other
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
Project description:Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.
| S-EPMC1178866 | biostudies-other