The reaction of diethyl pyrocarbonate with pyruvate kinase.
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ABSTRACT:
SUBMITTER: Dann LG
PROVIDER: S-EPMC1166129 | biostudies-other | 1974 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC1161179 | biostudies-other
Project description:1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.
| S-EPMC1177682 | biostudies-other
Project description:Diethyl pyrocarbonate (DEPC) has been primarily used as a residue-specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid-beta (A?) peptides and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determined the modification sites by electrospray ionization quadrupole time-of-flight tandem MS (ESI Q-TOF MS/MS). Our results indicate that five residues in A?1-42 are modified in the presence of 30-fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of A?1-42 promotes peptide aggregation, as monitored through the loss of soluble A?42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect A?1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of A?1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects A? from DEPC modification. Sequencing of obtained Cu(II)-protected A?1-16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human A? and to speculate about the role of His residues in these processes.
| S-EPMC7262909 | biostudies-literature
Project description:Exposure of junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor to the histidine-specific reagent diethyl pyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of ryanodine binding. The pH-dependence of the inactivation of ryanodine binding by DEPC and the reversal of this inactivation by hydroxylamine suggests the modification of histidine residue(s) by the reagent. Kinetic analysis of the time course of inactivation of ryanodine binding by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The degree of inactivation of ryanodine binding by DEPC was decreased when high NaCl concentrations were present in the modification medium. The binding affinities for ryanodine and Ca2+ were not altered by DEPC modification. This modification decreased the total ryanodine-binding sites. DEPC modification increased the Ca(2+)-permeability of the SR vesicles. A variety of bivalent cations prevented the DEPC inactivation of ryanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > Mg2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine binding. It is suggested that a histidine residue(s) in the ryanodine receptor is involved, either in the binding of Ca2+, or in a conformational change that may be required for Ca2+ binding to its binding site(s). This modification of the ryanodine receptor resulted in inactivation of ryanodine binding and activation of Ca2+ release.
| S-EPMC1138038 | biostudies-other
Project description: Not available
| EMPIAR-11729 | biostudies-other
Project description:1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75-1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [(14)C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the (14)C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.
| S-EPMC1164911 | biostudies-other
Project description:Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.
| S-EPMC1164739 | biostudies-other
Project description:Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PK deficiency. Here, we describe the identification and characterization of AG-348, an allosteric activator of PK that is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients with PK deficiency and ultimately lead to clinical benefit.
| S-EPMC5609468 | biostudies-literature
Project description:Pyruvate kinase deficiency (PKD) is the most common enzyme defect of glycolysis and an important cause of hereditary, nonspherocytic hemolytic anemia. The disease has a worldwide geographical distribution but there are no verified data regarding its frequency. Difficulties in the diagnostic workflow and interpretation of PK enzyme assay likely play a role. By the creation of a global PKD International Working Group in 2016, involving 24 experts from 20 Centers of Expertise we studied the current gaps in the diagnosis of PKD in order to establish diagnostic guidelines. By means of a detailed survey and subsequent discussions, multiple aspects of the diagnosis of PKD were evaluated and discussed by members of Expert Centers from Europe, USA, and Asia directly involved in diagnosis. Broad consensus was reached among the Centers on many clinical and technical aspects of the diagnosis of PKD. The results of this study are here presented as recommendations for the diagnosis of PKD and used to prepare a diagnostic algorithm. This information might be helpful for other Centers to deliver timely and appropriate diagnosis and to increase awareness in PKD.
| S-EPMC7344868 | biostudies-literature
Project description:Muscle type of pyruvate kinase (PKM) is one of the key mediators of the Warburg effect and tumor metabolism. Due to alternative splicing, there are at least 12 known isoforms of the PKM gene, of which PKM1 and PKM2 are two major isoforms with only a 23 amino acid sequenced difference but quite different characteristics and functions. It was previously thought the isoform switch from PKM1 to PKM2 resulted in high PKM2 expression in tumors, providing a great advantage to tumor cells. However, this traditional view was challenged by two recent studies; one study claimed that this isoform switch does not occur during the Warburg effect; the other study asserted that the isoform switch is tissue-specific. Here, we re-analyzed the RNA sequencing data of 25 types of human tumors from The Cancer Genome Atlas Data Portal, and confirmed that PKM2 was the major isoform in the tumors and was highly elevated in addition to the entire PKM gene. We further demonstrated that the expression level of PKM1 significantly declined even though there was substantially increased expression of the entire PKM gene. The proportion of PKM1 in total transcript variants also significantly declined in tumors but the proportion of PKM2 did not change accordingly. Therefore, we conclude that the isoform switch of PKM1 does indeed occur, but it switches to other isoforms rather than PKM2. Considering the change in the expression levels of PKM1, PKM2 and the entire PKM gene, we propose that the upregulation of PKM2 is primarily due to elevated transcriptional levels of the entire PKM gene, instead of the isoform switch.
| S-EPMC4349452 | biostudies-literature