scholarly journals Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces

1992 ◽  
Vol 281 (3) ◽  
pp. 601-605 ◽  
Author(s):  
S S Keskar ◽  
M B Rao ◽  
V V Deshpande
Keyword(s):  
Gas Phase ◽  
Active Site ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Aspartic Acid Residue ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Kinetics Of

The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

scholarly journals Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site

1991 ◽  
Vol 278 (1) ◽  
pp. 63-68 ◽  
Author(s):  
A E P Adang ◽  
W J Moree ◽  
J Brussee ◽  
G J Mulder ◽  
A van der Gen
Keyword(s):  
Active Site ◽  
Current Knowledge ◽  
Control Level ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
British Library ◽  
Glutathione S Transferase ◽  
Enzyme Active Site ◽  
Active Site Cysteine

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

scholarly journals Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis

1985 ◽  
Vol 232 (1) ◽  
pp. 37-42 ◽  
Author(s):  
D M Lowe ◽  
P K Tubbs
Keyword(s):  
Active Site ◽  
Control Mechanism ◽  
Cysteine Residue ◽  
Time Dependent ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Thioester Bond ◽  
Active Site Cysteine Residue

Succinyl-CoA (3-carboxypropionyl-CoA) inactivates ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) in a time-dependent manner, which is partially prevented by the presence of substrates of the enzyme. The inactivation is due to the enzyme catalysing its own succinylation. Complete inactivation corresponds to about 0.5 mol of succinyl group bound/mol of enzyme dimer. The succinyl-enzyme linkage appears to be a thioester bond and is probably formed with the active-site cysteine residue that is normally acetylated by acetyl-CoA. Succinyl-CoA binds to 3-hydroxy-3-methylglutaryl-CoA synthase with a binding constant of 340 microM and succinylation occurs with a rate constant of 0.57 min-1. Succinyl-enzyme breaks down with a half-life of about 40 min (k = 0.017 min-1) at 30 degrees C and pH 7 and is destabilized by the presence of acetyl-CoA and succinyl-CoA. A control mechanism is postulated in which flux through the 3-hydroxy-3-methylglutaryl-CoA cycle of ketogenesis is regulated according to the extent of succinylation of 3-hydroxy-3-methylglutaryl-CoA synthase.

scholarly journals Structural environment of an essential cysteine residue of xylanase from Chainia sp. (NCL 82.5.1)*

1996 ◽  
Vol 316 (3) ◽  
pp. 771-775 ◽  
Author(s):  
Mala RAO ◽  
Suvarna KHADILKAR ◽  
Kavita R. BANDIVADEKAR ◽  
Vasanti DESHPANDE
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Cysteine Residue ◽  
Yellow Colour ◽  
Streptomyces Species ◽  
Complete Inactivation ◽  
Structural Environment ◽  
Reporter Group

N-(2,4-Dinitroanilino)maleimide (DAM) reacts covalently with the thiol group of the xylanase from Chainia leading to complete inactivation in a manner similar to N-ethylmaleimide, but provides a reporter group at the active site of the enzyme. Increasing amounts of xylan offered enhanced protection against inactivation of the xylanase by DAM. Xylan (5 mg) showed complete protection, providing evidence for the presence of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvement of l mol of cysteine residue per mol of enzyme, as reported earlier [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The second-order rate constant for the reaction of DAM with the enzyme was 3.61×103 M-1·min-1. The purified xylanase was alkylated with DAM and digested with pepsin. The peptides were separated by gel filtration. The specific modified cysteinyl peptide was further purified by reverse-phase HPLC. The active-site peptide was located visually by its predominant yellow colour and characterized by a higher A340 to A210 ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly different from that of cysteinyl peptide derived from a xylanase from a thermotolerant Streptomyces species, but showed the presence of a conserved aspartic acid residue consistent with the catalytic regions of other glucanases.

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

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-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.

scholarly journals Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

Biochemistry ◽  
2018 ◽  
Vol 57 (38) ◽  
pp. 5513-5523 ◽  
Author(s):  
Matthias Fellner ◽  
Joel A. Rankin ◽  
Benoît Desguin ◽  
Jian Hu ◽  
Robert P. Hausinger
Keyword(s):  
Lactobacillus Plantarum ◽  
Active Site ◽  
Cysteine Residue ◽  
Active Site Cysteine ◽  
Active Site Cysteine Residue

scholarly journals Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols

2006 ◽  
Vol 398 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Jingmin Zeng ◽  
Rachael A. Dunlop ◽  
Kenneth J. Rodgers ◽  
Michael J. Davies
Keyword(s):  
Active Site ◽  
Cysteine Proteases ◽  
Cysteine Residue ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Active Site Cysteine ◽  
Reactive Aldehydes ◽  
Modified Proteins ◽  
Active Site Cysteine Residue

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259–269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine–lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

scholarly journals An active-site peptide from pepsin C

1971 ◽  
Vol 123 (1) ◽  
pp. 75-82 ◽  
Author(s):  
J. Kay ◽  
A. P. Ryle
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Ph Values ◽  
Complete Inactivation ◽  
Active Site Sequence

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.

Sign in / Sign up

Export Citation Format

Share Document