Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Intra- and intermolecular electron transfer processes in redox proteins

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.     

Oxaloacetate decarboxylase from Pseudomonas stutzeri: purification and characterization

Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.     

Subunit composition of oxaloacetate decarboxylase and characterization of the alpha chain as carboxyltransferase

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits alpha, beta, gamma with Mr 65 000, 34 000 and 12 000, respectively. On dodecylsulfate/polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high-performance liquid chromatography in a dodecylsulfate-containing buffer. Biotin was localized exclusively in the alpha chain. Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the alpha chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin-Sepharose. No beta or gamma chain were detectable in this alpha chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated alpha chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [1-14C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of Na+ ions. The ease with which the alpha chain was released from the membrane characterize this subunit as a peripheral membrane protein. The beta and gamma chain, on the other hand, stick so firmly in the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins. Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the alpha chain, yielding a polypeptide of Mr 51 000 which was devoid of biotin. Degradation of the beta chain required prolonged incubation periods and was markedly influenced by Na+ ions which had a protective effect against proteolysis. A proton is required in the decarboxylation of oxaloacetate and CO2 arises as primary product. The other alternative, i.e. generation of HCO3- with H2O as substrate, has been excluded.     

Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase beta subunit in the energy coupling mechanism of the Na+ pump.

The membrane-bound beta subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the beta subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild-type enzyme and the beta subunit mutants N373D, N373L, S382A, S382D, S382T, R389A, and R389D. In these studies, pH profiles, Na+ binding affinities, Hill coefficients, Vmax values and inhibition by Na+ was determined. A prominent result is the complete lack of oxaloacetate decarboxylase activity of the S382A mutant at Na+ concentrations up to 20 mm and recovery of significant activities at elevated Na+ concentrations (KNa approximately 400 mm at pH 6.0), where the wild-type enzyme is almost completely inhibited. These results indicate impaired Na+ binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na+ concentrations is uncoupled from the vectorial events of Na+ or H+ translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism, which includes Na+ binding to center I contributed by D203 (region IIIa) and N373 (helix VIII) and center II contributed by Y229 (helix IV) and S382 (helix VIII). These centers are exposed to the cytoplasmic surface in the carboxybiotin-bound state of the beta subunit and become exposed to the periplasmic surface after decarboxylation of this compound. During the countertransport of 2 Na+ and 1 H+ Y229 of center II switches between the protonated and deprotonated Na+-bound state.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Dissociation of the sodium-ion-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits

Oxaloacetate decarboxylase was reconstituted from the purified alpha subunit and a Triton X-100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin-Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the beta + gamma subunits, and fraction II consisted of the biotin-containing alpha subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated alpha and beta + gamma subunits. The Na+ transport activity was recovered, if a mixture of subunits alpha and beta + gamma was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins beta + gamma and completed by an attachment of the peripheral subunit alpha.     

Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.     

Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride.

Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.     

Pulsed EPR analysis of tartrate dehydrogenase active-site complexes.

Mn2+.tartrate dehydrogenase.substrate complexes have been examined by electron spin echo envelope modulation spectroscopy. The occurrence of dipolar interactions between Mn2+ and 2H on [2H]pyruvate and [4-2H]NAD(H) confirms that Mn2+ binds at the enzyme active site. The 2H signal arising from labeled pyruvate was lost if the sample was incubated at room temperature, indicating that the enzyme catalyzes exchange between the pyruvate methyl protons and solvent protons. Mn-133Cs dipolar coupling was also observed, which suggests that the monovalent cation cofactor also binds in the active site. The tartrate analogue oxalate was observed to have a significant effect on the binding of NAD(H). Oxalate appears to constrain the binding of NAD(H) so that the nicotinamide portion of the cofactor is held in close proximity to Mn2+. Spectra of enzyme complexes prepared with (R)-[4-2H]NADH showed a more intense 2H signal than analogous complexes prepared with (S)-[4-2H]NADH, demonstrating that the pro-R position of NADH is closer to Mn2+ than the pro-S position and suggesting that tartrate dehydrogenase is an A-side-specific dehydrogenase. Oxalate also affected Cs+ binding; the intensity of the 133Cs signal increased in the presence of oxalate, which suggest that oxalate facilitates binding of Cs+ to the active site or that Cs+ binds closer to Mn2+ when oxalate is present. In addition to signals from substrates, electron spin echo envelope modulation spectra revealed 14N signals that arose from coordination to Mn2+ by nitrogen-containing ligands from the protein; however, the identity of this ligand or ligands remains obscure.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Mechanisms of sodium transport in bacteria

In some bacteria, an Na+ circuit is an important link between exergonic and endergonic membrane reactions. The physiological importance of Na+ ion cycling is described in detail for three different bacteria. Klebsiella pneumoniae fermenting citrate pumps Na+ outwards by oxaloacetate decarboxylase and uses the Na+ ion gradient thus established for citrate uptake. Another possible function of the Na+ gradient may be to drive the endergonic reduction of NAD+ with ubiquinol as electron donor. In Vibrio alginolyticus, an Na+ gradient is established by the NADH: ubiquinone oxidoreductase segment of the respiratory chain; the Na+ gradient drives solute uptake, flagellar motion and possibly ATP synthesis. In Propionigenium modestum, ATP biosynthesis is entirely dependent on the Na+ ion gradient established upon decarboxylation of methylmalonyl-CoA. The three Na(+)-translocating enzymes, oxaloacetate decarboxylase of Klebsiella pneumoniae, NADH: ubiquinone oxidoreductase of Vibrio alginolyticus and ATPase (F1F0) of Propionigenium modestum have been isolated and studied with respect to structure and function. Oxaloacetate decarboxylase consists of a peripheral subunit (alpha), that catalyses the carboxyltransfer from oxaloacetate to enzyme-bound biotin. The subunits beta and gamma are firmly embedded in the membrane and catalyse the decarboxylation of the carboxybiotin enzyme, coupled to Na+ transport. A two-step mechanism has also been demonstrated for the respiratory Na+ pump. Semiquinone radicals are first formed with the electrons from NADH; subsequently, these radicals dismutate in an Na(+)-dependent reaction to quinone and quinol. The ATPase of P. modestum is closely related in its structure to the F1F0 ATPase of E. coli, but uses Na+ as the coupling ion. A specific role of protons in the ATP synthesis mechanism is therefore excluded.     

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.     

Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate

Phosphoenolpyruvate carboxykinase from chicken liver mitochondria and rat liver cytosol catalyzes the phosphorylation of alpha-substituted carboxylic acids such as glycolate, thioglycolate, and DL-beta-chlorolactate in reactions with absolute requirements for divalent cation activators. 31P NMR analysis of the reaction products indicates that phosphorylation occurs at the alpha-position to generate the corresponding O- or S-bridged phosphate monoesters. In addition, the enzymes catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyzes the bicarbonate-dependent phosphorylation of fluoride ion. The kappa cat values for these substrates are 20-1000-fold slower than the kappa cat for oxaloacetate. Pyruvate and beta-hydroxypyruvate are not phosphorylated, since the enzyme does not catalyze the enolization of these compounds. Oxalate, a structural analogue of the enolate of pyruvate, is a competitive inhibitor of phosphoenolpyruvate carboxykinase (Ki of 5 microM) in the direction of phosphoenolpyruvate formation. Oxalate is also an inhibitor of the chicken liver enzyme in the direction of oxaloacetate formation and in the decarboxylation of oxaloacetate. The chicken liver enzyme is inhibited by beta-sulfopyruvate, an isoelectronic analogue of oxaloacetate. The extensive homologies between the reactions catalyzed by phosphoenolpyruvate carboxykinase and pyruvate kinase suggest that the divalent cation activators in these reactions may have similar functions. The substrate specificity indicates that phosphoenolpyruvate carboxykinase decarboxylates oxaloacetate to form the enolate of pyruvate which is then phosphorylated by MgGTP on the enzyme.     

Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change

The (Na+ + K+)ATPase is inhibited by the bee venom polypeptide, melittin. KCl and NaCl protect the enzyme from melittin inhibition. Analysis of the K+ and Na+ protection against melittin inhibition suggested a kinetic model which was consistent with slowly reversible melittin binding, and mutually exclusive binding of melittin with K+ and Na+. Accordingly, in the absence of salt, the KI for melittin inhibition = 1.2 microM, and the protection by KCl occurs with a KA,KCl = 0.6 mM. The protection by NaCl occurs with a KA,NaCl = 15 mM. Melittin inhibition of enzyme activity is due to direct interactions with the (Na+ + K+)ATPase, as demonstrated by photolabeling with [125I]azidosalicylyl melittin, which labeled the alpha subunit, but not the beta subunit of the (Na+ + K+)ATPase. Melittin and KCl reduced the extent of labeling. In non-covalent binding studies using [125I]azidosalicylyl melittin, the stoichiometry of binding was 1.6 melittin per (Na+ + K+)ATPase. Ligand-induced conformational changes of FITC-labeled (Na+ + K+)ATPase were examined in the presence and absence of melittin. K+ alone or melittin alone caused a fluorescence intensity quenching consistent with formation of an E2 form of the enzyme. The NaCl-induced (E2----E1) fluorescence intensity changes were maximal when the enzyme was treated with K+. NaCl-induced fluorescence changes did not occur when the enzyme was treated with melittin in the absence of K+. However, when K+ was present before the addition of melittin, NaCl-induced fluorescence intensity increases were observed, which were dependent upon the concentration of K+ in the preincubation mixture. The results of the labeling and conformational studies support the kinetic model and suggest a mechanism for inhibition of ion pumps by (poly)peptides.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis.

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

Plant pyruvate dehydrogenase complex: analysis of the kinetic properties and metabolite regulation

The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent K_m values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD⁺, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD⁺, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD⁺. Among various metabolites tested, only hydroxypyruvate (K_i = 0.11 mM) and glyoxylate (K_i = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg²⁺? or Ca²⁺-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.