Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The highly purified form of phosphoenolpyruvate carboxykinase (PEPCK) contained 13 thiols (all in the reduced state) per 72 000 daltons. Modification of the enzyme with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) caused rapid formation of a cystine disulfide bridge and an even more rapid loss of enzymatic activity. Formation of the cystine bridge proceeded about 25 times faster than formation of the analogous intramolecular disulfide of dithiothreitol induced by Nbs2. o-Iodosobenzoate, Cd2+, and the 2,3-dimercapto-1-propanol complex of arsenite were potent, time-dependent, irreversible inhibitors of PEPCK. The inactivation by arsenite-2,3-dimercapto-1-propanol and o-iodosobenzoate was first order with respect to both time and inhibitor concentration. The sum of these data indicates the existence in PEPCK of a critical cysteine that is in a vicinal dithiol grouping with a second cysteine. PEP protected against cystine bridge formation induced by equimolar Nbs2 but not against the extent of inactivation. In the presence of PEP, the modification by Nbs2 of one cysteine/mol of enzyme (k = 1.2 X 10(6) M-1 min-1 at pH 7.2) caused nearly complete inactivation. Replacing the bulky 5-thio-2-nitrobenzoate moiety with cyanide did not result in any reactivation. This critical, cyanylated cysteine was determined to be 44% of the distance from the amino terminus.
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PMID:A vicinal dithiol containing an essential cysteine in phosphoenolpyruvate carboxykinase (guanosine triphosphate) from cytosol of rat liver. 72 3

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.
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PMID:Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde. 188 94

Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.
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PMID:Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase. 212 5

We have recently reported that the light-induced changes in the enzymatic and regulatory properties of maize leaf phosphoenolpyruvate carboxylase are attributed to the regulatory seryl phosphorylation of this C4-photosynthesis enzyme. In the present study, the darkform target enzyme was phosphorylated/activated in vitro by a maize leaf protein-serine kinase, and the 32P-labeled regulatory site phosphopeptide was purified from a tryptic digest by metal-ion affinity and reversed-phase chromatography. Automated Edman degradation analysis by covalent protein sequencing technology revealed that the amino acid sequence of this phosphoseryl peptide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg. This nonapeptide, which corresponds exactly to residues 13-21 in the deduced primary sequence of the maize leaf carboxylase, is far removed from recently identified active-site cysteine (Cys-553) and lysine (Lys-606) residues in the C-terminal region of the primary structure. Comparative analysis of the deduced N-terminal sequences of C3-, C4-, and Crassulacean acid metabolism (CAM)-leaf phosphoenolpyruvate carboxylases suggests that the motif of Lys/Arg-X-X-Ser is an important structural requirement of the C4- and CAM-leaf protein-serine kinases.
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PMID:Regulatory phosphorylation of serine-15 in maize phosphoenolpyruvate carboxylase by a C4-leaf protein-serine kinase. 214 63

Modification of phosphoenolpyruvate carboxylase with o-phthalaldehyde (OPA) resulted in rapid and irreversible inactivation exhibiting biphasic reaction kinetics. The kinetic analysis and correlation of spectral changes with activity indicated that inactivation by OPA results from the modification of two lysine and two cysteine residues per subunit of the enzyme. PEP plus Mg2+ offered substantial protection against modification. Some of the effectors also gave appreciable protection against modification indicating that the residues may be located at or close to the active site. Thus, the results indicate formation of two isoindoles showing the proximity of the essential lysine and cysteine residues at the active site.
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PMID:o-Phthalaldehyde as a probe in the active site of phosphoenolpyruvate carboxylase. 238 97

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.
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PMID:Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase. 253 43

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.
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PMID:A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase. 254 99

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.
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PMID:A histidine residue at the active site of avian liver phosphoenolpyruvate carboxykinase. 258 87

Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) specifically utilizes a guanosine or inosine nucleotide as a substrate, yet it does not share extended sequence homology with other GTP-binding proteins, and the molecular basis for its nucleotide specificity is not understood. In an effort to locate the enzyme's nucleotide-binding site, we have studied the interaction of cytosolic PEPCK from rat liver with the photoprobe 8-azidoGTP, which fulfills the criteria of a specific photoaffinity label for PEPCK. The photoprobe binds reversibly to the enzyme prior to modification and at low concentrations causes greater than 60% inactivation (Ki = 1.2 microM). GTP provides nearly complete protection against inactivation by 8-azidoGTP, whereas phosphoenolpyruvate and metal ions provide partial protection. In addition, the photoprobe is a substrate for the enzyme and has a Km similar to that for GTP. However, the extent of covalent modification by [32P]8-azidoGTP as measured by three independent techniques is significantly lower than the extent of enzyme inactivation. Further investigation of this anomaly has revealed that the loss in enzymatic activity is caused by modification of a critical cysteine residue in a reaction that does not terminate with covalent attachment of the photolabel. Quantitation of the total free thiols of modified PEPCK shows that 2 mol of cysteine is lost per mole of inactivated enzyme. These results indicate that the photoinactivation of PEPCK by 8-azidoGTP is caused by the formation of an intramolecular cystine disulfide bridge, thus providing evidence for the existence of a pair of proximal cysteine residues within the GTP-binding site. The interaction of cysteine residues with the reactive photogenerated derivatives of 8-azidopurines is discussed.
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PMID:Formation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5'-triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabeling. 261 Dec 26

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.
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PMID:Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP). 290 19


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