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T Nowak

Publications and source records attributed to T Nowak.

At least 91 records · Page 5Linked to original sources
PubMed1984

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes.

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.

Animals↗
PubMed1983

3-Mercaptopicolinate. A reversible active site inhibitor of avian liver phosphoenolpyruvate carboxykinase.

The inhibition of chicken liver phosphoenolpyruvate carboxykinase by 3-mercaptopicolinic acid (3-MP) has been investigated. Kinetic studies show 3-MP to be a noncompetitive inhibitor relative to all substrates and to the activator, Mn2+. EPR studies demonstrate that Mn2+ binding to the enzyme is unaffected by 3-MP. Proton relaxation rate studies demonstrate that 3-MP binds to the binary E X Mn complex with a KD of 0.5 X 10(-6) M and gives a ternary enhancement of 8.0. Additional proton relaxation rate studies detected formation of the quaternary complexes E X Mn X IDP X 3-MP, E X Mn X ITP X 3-MP, and E X Mn X CO2 X 3-MP. High resolution 1H nuclear relaxation rate studies suggest that 3-MP binds in close proximity to the activator cation, Mn2+, but not in the first coordination sphere. Active site models suggest that the 3-MP-binding site may partially overlap the phosphoenolpyruvate-binding site. The NMR studies, which detected formation of the quaternary E X Mn X 3-MP X phosphoenolpyruvate complex, also demonstrated that the binding of one of these ligands affects the interactions of the other ligand with E X Mn. Calorimetric studies of the E X Mn complex demonstrated that 3-MP causes an increase in the transition temperature midpoint without an increase in enthalpy. These results indicate that 3-MP causes a conformational change in the enzyme but does not increase the thermostability of the ternary complex. The experiments reported herein suggest that inhibition by 3-MP is due to specific and reversible binding within the active site of phosphoenolpyruvate carboxykinase.

Animals↗
PubMed1983

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: stereospecificity of proton transfer in the phosphorylation of enzyme I from (Z)-phosphoenolbutyrate.

The stereochemistry of the proton transfer in the reaction of phosphoenolbutyrate with enzyme I has been established. During the reaction of the pure Z isomer of this analogue of phosphoenolpyruvate with enzyme I, to yield phosphoenzyme I and 2-oxobutyrate, the substrate is protonated at C-3 from the 2re,3si face. This stereospecificity was established for the transfer of a proton to (Z)-phospho[3-D]enolbutyrate and for the transfer of a deuteron to (Z)-phospho[3-H]enolbutyrate. The E isomer of phosphoenolbutyrate is not a substrate for enzyme I. Accordingly, the reaction of phosphoenzyme I with 2-oxobutyrate yields exclusively the Z isomer of phosphoenolbutyrate, and only the pro-S proton at C-3 of 2-oxobutyrate is abstracted. A kinetic H/D isotope effect of 6.8 in this reaction demonstrates the rate-limiting nature of the proton-transfer step. The stereochemical analysis of 2-oxo[3(R)-H,D]butyrate and of 2-oxo-[3(S)-H,D]butyrate was carried out by using the pyruvate kinase catalyzed enolization of this compound. This enzymatic enolization, with phosphate as a cofactor, is rapid at neutral pH and is a highly stereospecific reaction: only the pro-R proton at C-3 of 2-oxobutyrate is exchanged with solvent. This reaction was also used to generate the pure 3R and 3S enantiomers of 2-oxo[3-H,D]butyrate. The degree of protonation/deuteration at C-3 of 2-oxobutyrate was detected from the fine structure of the methyl proton nuclear magnetic resonance signal.

Escherichia coli↗
PubMed1982

The purification, characterization, and activation of phosphoenolpyruvate carboxykinase from chicken liver mitochondria.

The enzyme phosphoenolpyruvate carboxykinase has been purified from chicken liver mitochondria. This purification includes a pseudo-affinity column step utilizing Sepharose 4B-blue dextran which binds the enzyme. The enzyme elutes with ITP to yield protein which is greater than 98% pure. The enzyme has Mr = 75,400 +/- 200 estimated by high speed sedimentation equilibrium and 70,500 +/- 500 estimated by reduced sodium dodecyl sulfate-polyacrylamide gels. The enzyme is abnormally retarded on molecular exclusion resins yielding low apparent molecular weight values. The amino acid analysis indicates that the enzyme has a high proline content and a high tryptophan content and contains 9 mol of cysteine/mol of enzyme. No disulfide bonds were detected. The extinction coefficient (epsilon 1% 280 = 16.5 +/- 0.1) reflects the high tryptophan content. The Svedberg coefficient (s20,w = 4.63 +/- 0.03 S) is consistent with a globular protein of Mr = 70,500-75,400. The activation of the enzyme was investigated by steady state kinetics. The carboxylation reaction has an activation energy of 17.6 kcal/mol. There is no requirement of a monovalent cation for activity. A thiol is necessary for maximal activity, although apparently not to reduce disulfide bonds within the enzyme. Incubation with dithiothreitol stabilizes enzymatic activity but beta-mercaptoethanol facilitates loss of activity. The kinetics of activation by Mn2+ was performed. The Ks value for phosphoenolpyruvate (300 microM) decreases to an apparent Km of 67 microM with increasing concentrations of Mn2+. The concentration of Mn2+ does not affect the interaction of HCO-3 with the enzyme, however. Analysis of data in terms of free IDP indicates that increasing Mn2+ decreases the Km of IDP but analysis as MnIDP indicates the Km,app of MnIDP is independent of the Mn2+ concentration. The enzyme interacts with Mn2+ with a KA = 67 microM and the Km,app decreases to a value of 8 microM with saturating substrates. The substrate analogue (Z)-3-fluorophosphoenolpyruvate is a good substrate for the reaction (Km = 30 microM) with 27% Vmax compared to P-enolpyruvate (Km = 180 microM). Except for 3-bromophosphoenolpyruvate, other analogues have shown weak competitive or noncompetitive inhibition. Potential analogues of oxalacetate (succinate, citrate, isocitrate, malate, and alpha-ketoglutarate) all elicit weak (greater than 15 mM) inhibition.

Amino Acids↗
PubMed1981

The role of cations in avian liver phosphoenolpyruvate carboxykinase catalysis. Activation and regulation.

The activation of the avian liver phosphoenolpyruvate carboxykinase catalyzed reversible decarboxylation of oxalacetate by Mn2+ has been studied. The Mn2+ facilitates the interaction of oxalacetate to the enzyme. At saturating ITP and oxalacetate concentrations, Mn2+ has a Km = 2 microM. The cation Mg2+ can substitute for Mn2+ with 50% Vmax in the oxalacetate decarboxylation and 2% Vmax in the oxalacetate formation. The Km for Mg2+ is 3 orders of magnitude greater than the Km of Mn2+, however. Of the other cations tested (Ca2+, Zn2+, Fe2+, and Cd2+), Co2+ was the only other cation found to activate the enzyme in both directions. In the presence of Mg2+, the enzyme is extremely sensitive to trace metal contaminants which can cause activation. At 1 mM Mg2+, 20 microM Mn2+ causes a 15-fold activation of activity. The apparent Km for Mn2+ (2 microM) at high concentrations of Mg2+ is the identical value calculated for free Mn2+. In a mixed metal (Mg2+ and Mn2+) assay, the Km values for phosphoenolpyruvate and for oxalacetate are independent of the concentration of Mg2+ but decrease upon an increase in Mn2+. The kinetic results demonstrate two roles for the divalent cations for activity. The cation forms a metal . nucleotide complex which serves as the substrate. The results indicate that MgITP is a better substrate than MnITP, but MnIDP is a better substrate than MgIDP. The cation also binds to the enzyme to form an enzyme . metal complex which is the active form of the enzyme. This cation functions to aid in the interaction of either oxalacetate or phosphoenolpyruvate to the enzyme. The activation by micromolar amounts of Mn2+ at millimolar concentrations of Mg2+ suggests that this enzyme, and thus the pathway of gluconeogenesis, can be modulated by changes in concentration of Mn2+ within mitochondria.

Animals↗
PubMed1981

Pyruvate kinase: activation by and catalytic role of the monovalent and divalent cations.

This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K+ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact, E. coli synthesize, depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4-8 A of the divalent cation activator and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg+2 is optimal for the physiological reaction, however, Co+2, Mn+2, and Ni+2 also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.

Animals↗
PubMed1981

Comparative inhibition of mitochondrial and cytosolic phosphoenolpyruvate carboxykinases by stereospecific substrate analogues.

Phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] was purified from the liver of several species; the mitochondrial enzyme and the cytosolic enzyme were separated from each other. The species included guinea pig, monkey, rat, and chicken. Each enzyme was assayed for inhibition by the substrate analogues E-2-phosphoenolbutyrate and Z-2-phosphoenolbutyrate. Each enzyme tested displayed the same stereospecificity: the E diastereoisomer was the more potent inhibitor than the Z. These results suggest an active site homology for all carboxykinases. The absolute values for Ki measured show that in almost every case the mitochondrial enzyme is more susceptible to inhibition by these analogues than is the cytosolic enzyme. The Ki values are one-fifth those for the mitochondrial enzymes. These results imply subtle differences in ligand interactions at the active sites of these enzymes.

Animals↗
PubMed1978

Structural changes at the active site of pyruvate kinase during activation and catalysis.

The distance between the obligatory monovalent and divalent cations at the active site of pyruvate kinase (rabbit muscle) has been used to monitor structural changes at various successive intermediates in the catalytic reaction and to relate structure and activity of substrate analogs. Determinations of distance were obtained from measurements of the longitudinal relaxation rate (1/T1) of the methyl protons of methyl ammonium ion, which was affected by the activating divalent cation Mn2+ in the various enzyme complexes. A frequency dependence of the 1/T1 effects with several complexes indicats that the observed relaxation rate changes are modulated by changes in the cation-cation distances. The results are interpreted as a sequence of structural changes at the active site which occur upon substrate binding, catalysis, and product release. Substrate analogs which are good analogs of phosphoenolpyruvate by kinetic criteria induce structural changes analogous to the substrate. An attempt is made to correlate the cation-cation distance changes to other structural changes reported for pyruvate kinase.

Animals↗