Quasi-elastic light scattering studies on pyruvate oxidase.

Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.). Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase. Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation. In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A. The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.     

Minimum requirements for protease activation of flavin pyruvate oxidase.

Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.     

Analysis of the Escherichia coli ribosome-ribosomal subunit equilibrium using pressure-induced dissociation

Hydrostatic pressure can be used to perturb the ribosome-ribosomal subunit equilibrium. We have used glutaraldehyde fixation and subsequent sucrose gradient analysis to determine the equilibrium concentrations of Escherichia coli 70 S, 50 S, and 30 S particles at pressures from 1 to 1400 atm. This method is shown to be sufficiently rapid and free of interfering ribosomal aggregation artifacts when performed at Mg2+ concentrations below 8 mM. We show directly that the E. coli ribosome is in equilibrium with its subunits and that the pressure-sensitive reaction is appropriately described by the expression: In Kp = ln K0 + (P delta V/RT), where Kp and K0 are the equilibrium constants at pressure P and 1 atm, respectively, and delta V is the change in molecular volume that occurs during the reaction. The method provides values for K0 under different conditions, and the effects of Mg2+ ion can be readily ascertained. K0 and delta V were also estimated by a method of fitting computer-generated sucrose gradient profiles to experimental profiles. Determination of delta H0, delta S0, and delta V0 at 5 mM Mg2+ are presented. The results are discussed in the context of previous thermodynamic studies of the E. coli ribosome.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

The dissociation of glucose oxidase by sodium n-dodecyl sulphate.

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.     

A single-step large-scale purification of pyruvate oxidase

Pyruvate oxidase is an Escherichia coli peripheral membrane flavoprotein which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. Pyruvate oxidase, like several other peripheral membrane enzymes, can be activated either by binding to lipid amphiphiles or by limited protease digestion. This paper reports a rapid and convenient method for effecting the large-scale purification of pyruvate oxidase from crude enzyme preparations using a Triton X-114 phase separation technique. It appears likely that this purification procedure can be used successfully with the family of enzymes which respond to both lipid and protease activation.     

Conformational studies of Escherichia coli pyruvate oxidase

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.     

Binding of pyruvate oxidase alpha-peptide to phospholipid vesicles

The alpha-peptide of pyruvate oxidase is a 23 residue peptide which is cleaved from the carboxy terminus of the enzyme during proteolytic activation by chymotrypsin (M. Recny et al. (1985) J. Biol. Chem. 260, 14287-14291). Cleavage of alpha-peptide results in the loss of the high affinity lipid-binding site in the enzyme. The beta-peptide of pyruvate oxidase is a 101 residue peptide which also is cleaved from the carboxy terminus of pyruvate oxidase. Cleavage of the beta-peptide from pyruvate oxidase results in the inactivation of the enzyme. The beta-peptide includes the alpha-peptide amino acid sequences at its carboxyl terminus. We now report on the binding of the alpha- and beta-peptides to phospholipid vesicles. Both peptides bind with equal and high affinity to phosphatidylcholine vesicles. We conclude from these results that the alpha-peptide furnishes the membrane-binding site which plays the physiologically important role in the activation of this peripheral membrane enzyme.     

Determination of inorganic phosphorus using immobilized pyruvate oxidase

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one-month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well-established method.     

Enhanced dissociation of pyruvate dehydrogenase from the pyruvate dehydrogenase complex following phosphorylation and regulatory implications

We have shown that the active form of the pyruvate dehydrogenase (PDH_a) component exhibits at least a 9-fold greater affinity for sites on the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex than does the inactive (phosphorylated) form of pyruvate dehydrogenase (PDH_b). Consistent with a higher rate of dissociation for PDH_b than for PDH_a, free PDH_a rapidly replaces PDH_b whereas, even at high levels, free PDH_b only slowly replaces PDH_a. Dissociation of newly formed PDH_b, during phosphorylation by the immobile PDH_a kinase, leads to an increased association of free PDH_a as observed by protection against inactivation of the complex, even though PDH_a kinase activity is increased.     

Physiological role of pyruvate oxidase in the aerobic metabolism of Lactobacillus plantarum.

Under aerobic growth conditions Lactobacillus plantarum produced acetic acid in addition to lactic acid. It was found that lactic acid was predominantly produced at first, and then when the carbohydrate was nearly exhausted, lactic acid was metabolized further to acetic acid. The most likely enzyme involved in the aerobic metabolism of L. plantarum is pyruvate oxidase. Its activity is enhanced in the presence of oxygen and is reduced in the presence of glucose. The specific activity of pyruvate oxidase is highest at the beginning of the stationary-growth phase, where a strong increase in acetic acid production was also observed.     

The pressure-induced inactivation of mammalian enolases is accompanied by dissociation of the dimeric enzyme

The effects of exposure to pressure on both the activity and the quaternary structure of rabbit brain enolases, forms alpha alpha, alpha gamma, and gamma gamma were studied in the pressure range of 1 to 3400 bar. Effects on quaternary structure were determined by subunit scrambling (the formation of alpha alpha and gamma gamma from alpha gamma or vice versa). All three dimers are stable up to pressures of 1200 bar. The dissociation of gamma gamma begins at 1200 bar, yielding a stable monomer; inactivation of gamma gamma does not begin until the pressure is greater than 2000 bar. Dissociation of gamma gamma is not accompanied by changes in the tryptophan fluorescence of the protein. However, the fluorescence does decrease when the pressure is greater than 2000 bar, the point at which inactivation of gamma gamma starts. The alpha monomer, on the other hand, is unstable in the pressure range that produces dissociation of alpha alpha. This process, which also begins at 1200 bar, is paralleled by inactivation. Crosslinking the enzyme with glutaraldehyde demonstrated that the inactive form of the enzyme is monomeric. The pressure-induced inactivation of these forms of enolase is thus clearly a two-step process, with both dissociation and inactivation occurring. The difference in pressure sensitivity of rabbit brain alpha alpha and gamma gamma is due to a difference in stability of the alpha and gamma monomers and not due to a difference in the pressures required for dissociation.