scholarly journals The electrostatic fields in the active-site clefts of actinidin and papain

1988 ◽  
Vol 254 (1) ◽  
pp. 235-238 ◽  
Author(s):  
R W Pickersgill ◽  
P W Goodenough ◽  
I G Sumner ◽  
M E Collins
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Structural Similarity ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Activity Profile ◽  
Active Site Cysteine ◽  
Electrostatic Fields ◽  
Imidazolium Ion ◽  
Active Site Cysteine Residue

The active sites of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2) display different reactivity characteristics to probes targeted at the active-site cysteine residue despite the close structural similarity of their active sites. The calculated electrostatic fields in the active-site clefts of actinidin and papain differ significantly and may explain the reactivity characteristics of these enzymes. Calculation of electrostatic potential also focuses attention on the electrostatic properties that govern formation of the active-site thiolate-imidazolium ion-pair. These calculations will guide the modification of the pH-activity profile of the cysteine proteinases by site-directed mutagenesis.

scholarly journals Recombinant expression and isolation of human l-arginine:glycine amidinotransferase and identification of its active-site cysteine residue

1997 ◽  
Vol 322 (3) ◽  
pp. 771-776 ◽  
Author(s):  
Andreas HUMM ◽  
Erich FRITSCHE ◽  
Karlheinz MANN ◽  
Martin GÖHL ◽  
Robert HUBER
Keyword(s):  
Active Site ◽  
Recombinant Expression ◽  
Factor Xa ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cleavage Sites ◽  
Fusion Peptides ◽  
Kidney Mitochondria ◽  
Active Site Cysteine ◽  
Active Site Cysteine Residue

Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine: glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coliwith two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

scholarly journals Evidence for histidine in the active sites of ficin and stem-bromelain

1968 ◽  
Vol 110 (1) ◽  
pp. 53-57 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Active Site Cysteine ◽  
Stem Bromelain ◽  
Hydrolysis Of ◽  
Active Site Cysteine Residue

1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5Å locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only.

scholarly journals 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

1996 ◽  
Vol 320 (1) ◽  
pp. 181-186 ◽  
Author(s):  
David COMMUNI ◽  
Christophe ERNEUX
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type I ◽  
Active Site Cysteine ◽  
Substrate Binding Domain ◽  
Active Site Cysteine Residue

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 iodoacetamide. 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 α-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.

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.

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