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

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.

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

Biochemical Journal ◽

10.1042/bj2810601 ◽

1992 ◽

Vol 281 (3) ◽

pp. 601-605 ◽

Cited By ~ 7

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.

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Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

Biochemical Journal ◽

10.1042/bj3081001 ◽

1995 ◽

Vol 308 (3) ◽

pp. 1001-1007 ◽

Cited By ~ 21

Author(s):

M P Chadwick ◽

F E B May ◽

B R Westley

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Gel Filtration ◽

Thiol Group ◽

Cysteine Residue ◽

Exchange Chromatography ◽

Single Domain ◽

Trefoil Peptides ◽

Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

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

Biochemical Journal ◽

10.1042/bj2780063 ◽

1991 ◽

Vol 278 (1) ◽

pp. 63-68 ◽

Cited By ~ 13

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.

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

Biochemical Journal ◽

10.1042/bj2320037 ◽

1985 ◽

Vol 232 (1) ◽

pp. 37-42 ◽

Cited By ~ 44

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.

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NRPS condensation domain–substrate studies using X-ray crystallography

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095618 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C438-C438

Author(s):

Kristjan Bloudoff ◽

Thomas Schmeing

Keyword(s):

Active Site ◽

Streptomyces Coelicolor ◽

Covalent Bond ◽

Thiol Group ◽

Peptide Bond ◽

Cysteine Residue ◽

Local Concentration ◽

Peptide Bond Formation ◽

Nonribosomal Peptide ◽

Condensation Domain

Nonribosomal peptide synthetases (NRPSs) are a family of large multimodular enzymes that synthesize structurally and functionally diverse peptides including siderophores, toxins, agriculturally-important compounds and pharmaceutically-important compounds. The condensation (C) domain is responsible for peptide bond formation, the central chemical step in nonribosomal peptide synthesis. Here we present the crystal structure of the first condensation domain of the calcium-dependent antibiotic (CDA) synthetase (CDA-C1) from Streptomyces coelicolor soaked with a small molecule compound representing the acceptor substrate. To increase the likelihood of complex formation, we designed the compound to contain a free thiol group in order to form a covalent bond between the substrate and a cysteine residue of the CDA-C1. The tethering of the substrate to the active site mimics delivery of substrate to the active site by the NRPS PCP domain, and the disulfide bond it forms with the protein will ensure a high local concentration of substrate. Initial maps, calculated from diffraction datasets collected at the home source, indicated density corresponding to the presence of substrate at the active site. This result, along with activity assay data, will help implicate residues important to enzyme catalysis and substrate specificity. In all, these studies will help characterize C domain function in NRPSs and potentiate the use of NRPSs in bioengineering experiments to produce novel or improved therapeutics.

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

Biochemical Journal ◽

10.1042/bj2610431 ◽

1989 ◽

Vol 261 (2) ◽

pp. 431-435 ◽

Cited By ~ 16

Author(s):

H G Nimmo ◽

F Douglas ◽

C Kleanthous ◽

D G Campbell ◽

C MacKintosh

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Isocitrate Lyase ◽

Cysteine Residue ◽

Order Process ◽

Reactive Group ◽

Site Isolation ◽

First Order ◽

Complete Inactivation ◽

Ph Range

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.

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Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent

Biochemical Journal ◽

10.1042/bj2420499 ◽

1987 ◽

Vol 242 (2) ◽

pp. 499-503 ◽

Cited By ~ 20

Author(s):

C G Sanny ◽

H Weiner

Keyword(s):

Catalytic Activity ◽

Aldehyde Dehydrogenase ◽

Active Site ◽

Active Sites ◽

Specific Activity ◽

Cysteine Residue ◽

Complete Inactivation ◽

Horse Liver ◽

Inactivation Mechanism ◽

Liver Aldehyde Dehydrogenase

The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. The number of functioning active sites per tetrameric enzyme, as determined by the magnitude of the pre-steady-state burst of NADH formation, did not decrease until approx. 75% of the catalytic activity was lost. These data indicate that disulfiram does not modify the essential nucleophilic amino acid at the active site of the enzyme. The data support an inactivation mechanism involving the formation of a mixed disulphide with a non-essential cysteine residue, resulting in a lowered specific activity of the enzyme.

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A dramatic change in the rate-limiting step of β-lactam hydrolysis results from the substitution of the active-site serine residue by a cysteine in the class-C β-lactamase of Enterobacter cloacae 908R

Biochemical Journal ◽

10.1042/bj2920537 ◽

1993 ◽

Vol 292 (2) ◽

pp. 537-543 ◽

Cited By ~ 9

Author(s):

A Dubus ◽

D Monnaie ◽

C Jacobs ◽

S Normark ◽

J M FrÉre

Keyword(s):

Active Site ◽

Enterobacter Cloacae ◽

Order Rate Constant ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Serine Residue ◽

Rate Limiting Step ◽

Class C ◽

Rate Limiting ◽

Selective Influence

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by site-directed mutagenesis. The modified protein exhibited drastically reduced kcat./Km values on all tested substrates. However, this decrease was due to increased Km values with some substrates and to decreased kcat. values with others. These apparently contradictory results could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypothesis which was confirmed by the absence of detectable acylenzyme accumulation with all the tested substrates, with the sole exception of cefoxitin.

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Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group

Biochemical Journal ◽

10.1042/bj3000025 ◽

1994 ◽

Vol 300 (1) ◽

pp. 25-30 ◽

Cited By ~ 16

Author(s):

T M Kitson ◽

K E Kitson

Keyword(s):

Aldehyde Dehydrogenase ◽

Active Site ◽

Negative Charge ◽

Positive Charge ◽

Gel Filtration ◽

Modified Enzyme ◽

Covalently Linked ◽

Reporter Group

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (‘DMNB’) reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

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Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653442 ◽

1981 ◽

Vol 46 (03) ◽

pp. 658-661 ◽

Cited By ~ 112

Author(s):

C Korninger ◽

J M Stassen ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Intravenous Injection ◽

Active Site ◽

Half Life ◽

Degradation Products ◽

Gel Filtration ◽

Tissue Type ◽

Organ Distribution ◽

Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

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