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PubMed1981

The inactivation of thymidylate synthase by diethyl pyrocarbonate.

Thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) from Lactobacillus casei was inactivated by treatment with diethyl pyrocarbonate. The inactivation was apparently due to the modification of a large proportion of the enzyme's histidine residues. Neither the substrate dUMP nor the product dTMP prevented inactivation by diethyl pyrocarbonate. The inactivated enzyme was not reactivated by treatment with hydroxylamine. These results indicate that histidine residues are involved in the maintenance of enzyme structure.

Chemical Phenomena↗

PubMed1978

Inhibition of ribonuclease. Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, protein ase K and heparin using a sensitive ribonuclease assay.

The effectiveness of several commonly used inhibitors of ribonuclease (RNAase) has been studied using the removal of radio-labelled leucine from leucyl-tRNA as a sensitive assay for RNAase activity. The inhibitors were tested under a variety of conditions, varying the temperature, the pH, and the source of RNAase. When each inhibitor is udes separately in the presence of pancreatic RNAase, sodium dodecyl sulfate (SDS) is the most effective; but during long exposures to temperatures above 0 degrees C considerable amounts of RNA are still degraded. Combination of inhibitors are more effective in preserving RNA; with this assay, a combination of SDS with diethyl pyrocarbonate is the most effective. Proteinase K acts as an inhibitor when used in combination with SDS; however, it has RNAase activity when used by itself. Diethyl pyrocarbonate, when used at the high range of concentrations employed by others for RNAase inhibition, reacts with RNA changing its charge. However, when diethyl pyrocarbonate is used in smaller amounts the effects on RNA are minimal, and when used in combination with SDS it effectively inhibits RNAase.

Diethyl Pyrocarbonate↗

PubMed1969

Polyribosome isolation in the presence of diethyl pyrocarbonate.

Isolation of polyribosomes from wheat embryos and corn root tips in the presence of diethyl pyrocarbonate showed this reagent to have a protective effect on polyribosome structure. In addition, the use of diethyl pyrocarbonate allowed initial homogenization to be performed under less stringent conditions than those normally employed for polyribosome isolation. The use of the reagent is however limited, in that it is deleterious to in vitro ribosomal amino acid incorporation.

Amino Acids↗

PubMed1986

Diethyl pyrocarbonate: a chemical probe for DNA cruciforms.

Two palindromic DNA sequences were analyzed with respect to their chemical reactivities with diethyl pyrocarbonate. In negatively supercoiled plasmid templates enhanced N7 carbethoxylation was found with individual purines located in presumptive single-stranded loops of DNA cruciform structures. No enhanced reactivity at these positions was observed in linear, relaxed or low superhelical density plasmids. Hyperreactivity was found over a narrow region only, indicating that stable cruciforms contain loops of minimal size. No enhanced chemical reactivity was found with the four-way junction at the base of cruciforms. Diethyl pyrocarbonate has proved a sensitive structural probe for the analysis, with single nucleotide resolution, of DNA cruciform structures.

Base Sequence↗

PubMed1990

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

Binding Sites↗

PubMed1979

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate.

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate was studied. It was found that up to five of the sixteen histidine groups per enzyme subunit could be modified, and under the conditions employed, there was no evidence for formation of the disubstituted derivative of histidine. Evidence was obtained for small but significant amounts of modification of lysine and cysteine groups; tyrosine groups were not modified. Modification of the enzyme led to inactivation; this could be protected against by inclusion of substrates or, more effectively, by inclusion of the combination MgADP plus creatine plus nitrate, which is thought to produce a 'transition-stage-analogue' complex. Analysis of data on the rates of inactivation and the stoicheiometry of modification suggested that there was one essential histidine group per enzyme subunit, modification of which led to inactivation.

Adenosine Diphosphate↗

PubMed1988

Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding.

Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

Bacteriophage lambda↗

PubMed1991

Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli.

The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.

Amino Acid Sequence↗

PubMed1986

Diethyl pyrocarbonate (an imidazole binding substance) inhibits rostral VLM CO2 sensitivity.

Diethyl pyrocarbonate (DEPC) has been useful in vitro as an agent relatively specific for binding to imidazole of histidine. Administered via the cisterna magna DEPC inhibits central chemosensitivity in conscious rabbits, supporting the alphastat hypothesis for central chemoreceptor function. In this study I have applied DEPC via 1 X 3 mm cottonoid pledgets to each of the three ventrolateral medulla (VLM) chemosensitive areas in glomectomized, vagotomized, paralyzed, and servo-ventilated alpha-chloralose-urethan-anesthetized cats. CO2 responses measured by integrated phrenic nerve output were evaluated before and after DEPC application. A dose of 40 mmol/l applied to the rostral chemosensitive area increased the CO2 threshold (5.3%) and significantly decreased (P less than 0.03; Wilcoxon sign rank test) the initial slope (-43%) and the maximum (-41%) of the CO2 response. No significant effects were observed with DEPC application in the intermediate or caudal areas. Treatment with 40 mmol/l hydroxylamine immediately after DEPC in the rostral area prevented the effects supporting the interpretation that imidazole was the reactant with DEPC. The results are consistent with the hypothesis that imidazole-histidine is involved in the mechanism of central chemoreception and indicate that only the rostral area utilizes a DEPC inhibitable mechanism.

Animals↗

PubMed1981

Inhibition of D(--)-beta-hydroxybutyrate dehydrogenase by butanedione, phenylglyoxal, and diethyl pyrocarbonate.

D(--)-beta-Hydroxybutyrate dehydrogenase of beef heart mitochondria is inhibited by modifiers of disulfides, thiols, and vicinal dithiols [Phelps, D. C., & Hatefi, Y. (1981) Biochemistry 20 (preceding paper in this issue)]. The vicinal dithiol can be reversibly oxidized by diamide, resulting in activity inhibition, and rereduced by dithiothreitol, resulting in reactivation. The diamide-treated enzyme can no longer be irreversibly inhibited by N-ethylmaleimide, indicating the absence of an essential sulfhydryl group other than the vicinal dithiol. beta-Hydroxybutyrate dehydrogenase also appears to contain essential arginyl residues modifiable by phenylglyoxal or butanedione, and essential residue(s) modifiable at pH 6.0 by diethyl pyrocarbonate. Substrates protect against inhibitions by butanedione, phenylglyoxal, and diethyl pyrocarbonate, suggesting that the essential, modifiable residues are at or near the substrate binding sites. On the basis of these results and pH profiles, tentative mechanisms have been proposed for the oxidation of beta-hydroxybutyrate and the reduction of acetoacetate, involving the participation of the essential residues described above.

Acetoacetates↗

PubMed1984

Specific inactivation of the phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system by diethyl pyrocarbonate.

We have examined the interactions of the histidine-specific reagent diethyl pyrocarbonate (DEPC) with the components of the rat hepatic glucose-6-phosphatase system (EC 3.1.3.9). DEPC is the first known reagent that satisfies the criteria of an active-site-specific label for the phosphohydrolase component. (a) It inactivates through formation of a stable covalent bond. (b) It is effective at reasonably low concentrations (2-4 mM) under relatively mild conditions (e.g. 30 degrees C at neutral pH). (c) Inactivation is substantially blocked by glucose 6-phosphate, Pi and NaF, compounds which are known to interact quite specifically with the phosphohydrolase. (d) Under conditions where glucose 6-phosphate and NaF protect the enzyme, no protection is provided against DEPC-mediated inactivation of two other functional components of the membrane, the glucose 6-phosphate translocase and UDP-glucuronyltransferase. DEPC also shows potential for use at 0 degree C as a label for UDP-glucuronyltransferase.

Animals↗

PubMed1990

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase.

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.

Adenosine Triphosphatases↗

PubMed2001

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites.

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

Cytochrome b Group↗

PubMed1994

Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.

Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.

Affinity Labels↗

PubMed1995

The pH dependence and modification by diethyl pyrocarbonate of isocitrate lyase from Phycomyces blakesleeanus.

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaxpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units. Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second-order rate constant of 18.58 M-1 s-1 (at pH 6.0 and 20 degrees C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N-carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O-carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N-carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data-protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.

Binding Sites↗

PubMed1985

In vivo formation of a carcinogenic substance from diethyl pyrocarbonate in the presence of ammonia.

Mice of different strains have been treated repeatedly with diethyl pyrocarbonate (DEPC) and/or ammonia by gastric tube. In adult mice treated with DEPC plus ammonia, pulmonary tumours developed. However, DEPC or ammonia alone proved not to have any carcinogenic effect. When DEPC administration was followed by ammonia treatment more pulmonary tumours developed than in the case of ammonia-DEPC sequence. The shorter the time interval between DEPC and ammonia administrations the higher the number of lung tumours observed. Pulmonary tumours could not be observed in the offspring of pregnant mice treated with DEPC and ammonia or in suckling mice whose mother was treated with DEPC and ammonia. In gastric juice a new substance is formed from DEPC in the presence of ammonia. This new substance was very similar or identical to urethane according to the thin layer chromatographic investigation.

Ammonia↗

PubMed1988

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation.

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

Carbohydrates↗

PubMed1996

Modification of tomato and Aspergillus niger pectinesterases with diethyl pyrocarbonate.

The role of histidine residues in pectinesterases was evaluated by monitoring the sensitivity to modification with diethyl pyrocarbonate in the tomato and Aspergillus niger enzymes. Different and incomplete losses of enzyme activity were obtained. Inactivation of the enzymes was proportional to the histidine content (two in the tomato T1 form, six in the Aspergillus form), suggesting that accessible histidine residues do not have active-site functions in these pectinesterases, but contribute to the overall structural stability. Lack of His roles in common between the enzyme forms is in agreement with the structures of pectinesterases having no conserved His residues.

Aspergillus niger↗