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Amino acid sequence of a peptide containing the active cysteine residue of histidine ammonia-lyase.

ABSTRACT: 1. Oxidized (polymerized) histidine ammonia-lyase from Pseudomonas testosteroni was activated with dithiothreitol and the reduced disulphide-linked cysteine residues of the native enzyme were carboxymethylated with iodo[(14)C]acetate. 2. The activity of the carboxymethylated enzyme was similar to that of the polymerized form and approx. 15% of that of the fully reduced form. 3. A tryptic digest of the [(14)C]carboxymethylated enzyme contained only one radioactive peptide. 4. The amino acid sequence of this peptide was shown to be Gly-Leu-Leu-Asp-Gly-Ser-Ala-Ile-Asn-Pro-Ser-His-Pro-Asn-Cys- (CH(2)CO(2)H)-Gly-Arg. 5. These findings show that, during polymerization, the disulphide bonds are formed between identical regions of the enzyme, and that the cysteine residue involved is also the one required in the reduced state for full activity of the enzyme.

SUBMITTER: Hassall H

PROVIDER: S-EPMC1166157 | biostudies-other | 1974 Mar

REPOSITORIES: biostudies-other

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The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

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

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

Project description:The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.

| S-EPMC208851 | biostudies-other

The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain.

Project description:Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-(14)C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and alpha-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin.

| S-EPMC1178867 | biostudies-other

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

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

The location of the active-site histidine residue in the primary sequence of papain.

Project description:Papain that had been irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and alpha-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

| S-EPMC1198893 | biostudies-other

The amino acid sequence around the reactive cysteine residue in human plasma Factor XII.

Project description:1. Activated human plasma factor XIII was 65% inhibited by iodo[(14)C]acetate and incorporated 0.6 mol of label into the alpha subunit, which eventually was allowed to form a precipitate. 2. All the label was recovered as S-carboxymethylcysteine in a tetra-peptide of sequence Gly-Gln-Cys-Trp.

| S-EPMC1165912 | biostudies-other

The roles of active site residues in the catalytic mechanism of methylaspartate ammonia-lyase.

Project description:Methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l-threo-(2S,3S)-3-methylaspartate and l-erythro-(2S,3R)-3-methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys-331 acts as the (S)-specific base catalyst and abstracts the 3S-proton from l-threo-3-methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg(2+) ion and hydrogen bonding to the Gln-329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His-194 likely acts as the (R)-specific base catalyst and abstracts the 3R-proton from the l-erythro isomer of 3-methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site-directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio- and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL-catalyzed reaction, in which the side chains of Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.

| S-EPMC3722577 | biostudies-literature

Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

Project description:In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

| S-EPMC3640076 | biostudies-literature

pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

Project description:Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.

| S-EPMC6385126 | biostudies-literature

The chemical reactivity of the histidine-195 residue in lactate dehydrogenase thiomethylated at the cysteine-165 residue.

Project description:The specific thiomethylation of cysteine-165 (insertion of a methylthio group, CH3-S-) in pig heart lactate dehydrogenase results in a decreased affinity for carbonyl ligands that is accompanied by a decreased nucleophilic reaction of histidine-195 with diethyl pyrocarbonate. The rate constants at 10 degrees C for the modification of native and thiomethylated lactate dehydrogenase by diethyl pyrocarbonate were 173 M-1 . s-1 and 8.7 M-1 . s-1 respectively. It was found that 0.86 +/- 0.07 histidine residue per subunit reacted with diethyl pyrocarbonate in thiomethylated lactate dehydrogenase. This reaction was not affected in the enzyme-NADH binary complex, but was diminished in the enzyme-NADH-oxamate ternary complex. In the enzyme-NADH complex the reaction of diethyl pyrocarbonate was controlled by two groups with pKa 6.8 and 7.9. The decreased reactivity of histidine-195 was selective in thiomethylated lactate dehydrogenase, since the reactivity of arginine and/or lysine residues was enhanced.

| S-EPMC1162580 | biostudies-other

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