Effect of Physical Factors on Reactions of Horse-Radish Peroxidase Complexes with Reduced Cytochrome c

This investigation concerns the effect of certain physical factors—viscosity, dielectric constant, ionic strength, and temperature of the medium—on the reaction of hydrogen peroxide and ferrocytochrome c in the presence of the enzyme horse-radish peroxidase. From study of the effects of viscosity and dielectric constant, it was concluded that the reaction between the secondary complex of hydrogen peroxide and enzyme on the one hand and ferrocytochrome c on the other is controlled by diffusion in media of high viscosity and by electrostatic effects at low viscosities. With respect to ionic strength, the data at pH 4.7 indicated a dipole-dipole interreaction. The temperature dependence of the over-all reaction had a Q₁₀ of 1.25.     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate and by metal ions

The lactate dehydrogenase from Streptococcus faecalis is activated either by fructose 1,6-bisphosphate or by divalent cations such as Mn2+ or Co2+. With both types of activator, a lag is observed before attainment of the steady state rate of pyruvate reduction if the activator is added to the enzyme at the same time as the substrates. This lag can be largely abolished by preincubation of enzyme with activator before mixing with substrates. For fructose 1,6-bisphosphate (Fru(1,6)P2) as the activator, the rate constant for the lag phase showed a linear dependence on activator concentration but was independent of enzyme concentration. This suggests that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme which leads to increased activity, without association of enzyme subunits or dimers. With Co2+ as activator, the rate constant for the lag phase showed a hyperbolic dependence on Co2+ concentration and was also dependent on enzyme concentration. This suggests that activation by Co2+, in contrast to that by Fru(1,6)P2, involves association of enzyme dimers, followed by ligand binding.     

Thermodynamic and kinetic characterization of the association of triosephosphate isomerase: the role of diffusion

It is known that diffusion plays a central role in the folding of small monomeric proteins and in the rigid-body association of proteins, however, the role of diffusion in the association of the folding intermediates of oligomeric proteins has been scarcely explored. In this work, catalytic activity and fluorescence measurements were used to study the effect of viscosity in the unfolding and refolding of the homodimeric enzyme triosephosphate isomerase from Saccharamyces cerevisiae. Two transitions were found by equilibrium and kinetic experiments, suggesting a three-state model with a monomeric intermediate. Glycerol barely affects DeltaG(0)(fold) whereas DeltaG(0)(assoc) becomes more favourable in the presence of the cosolvent. From 0 to 60% (v/v) glycerol, the association rate constant showed a near unitary dependence on solvent viscosity. However, at higher glycerol concentrations deviations from Kramers theory were observed. The dissociation rate constant showed a viscosity effect much higher than one. This may be related to secondary effects such as short-range glycerol-induced repulsion between monomers. Nevertheless, after comparison under isostability conditions, a slope near one was also observed for the dissociation rate. These results strongly suggest that the bimolecular association producing the native dimer is limited by diffusional events of the polypeptide chains through the solvent.     

Agitation induced cell injury in microcarrier cultures. Protective effect of viscosity is agitation intensity dependent: Experiments and modeling

The effect of medium viscosity on the specific death rate of bovine embryonic kidney (BEK) cells cultured in spinner flask microcarrier cultures has been examined for various impeller speeds. Two types of media were used, a serum-containing growth medium and a serum-free maintenance medium. The latter does not support cell growth. We found that increasing medium viscosity suppresses cell death rates in both growth and maintenance medium cultures in an agitation-intensity-dependent fashion; the beneficial effect of medium viscosity in reducing the specific death rate is amplified as the agitation rate is increased. Furthermore, increasing medium viscosity has no effect on the specific death rate of the cells when the agitation rate is below a critical level. A model based on the turbulent energy content of eddies in the dissipation spectrum of turbulence of length scales on the order of magnitude of the microcarrier diameter and lower has been developed to account for cell death due to both bead-to-bead and bead-to-eddy interactions. The model constitutes a significant departure from previous efforts first because both types of interactions are accounted for simultaneously and second because the properties of a spectrum of eddies instead of the Kolmogorov-scale eddy size alone are used in the model. The model explains the functional dependence of the specific death rates on the medium viscosity at varying agitation intensities.     

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.     

Dependence on surface pH and surface substrate concentration of activity of microsome-bound arylsulfatase C and the surface charge density in the vicinity of the enzyme

Dependence on the salt concentration of the activity of microsome-bound arylsulfatase C [EC 3.1.6.1] from rat liver was examined. The activity increased with increasing salt concentration in the reaction medium in the whole pH range tested. This effect can be explained by the dependence of the reaction rate on the surface pH and the surface concentration of the ionic substrate. The dependence on salt concentration of the activity of the microsome-bound arylsulfatase C and the pH-dependences of Vmax and Km of the enzyme were used for the estimation of pH at the microsomal surface. The two values of the surface pH (surface potential) and the salt concentration were applied to the Gouy-Chapman equation. The value of -0.39 +/- 0.08 X 10(-3) elementary charge/A2 was obtained as the surface charge density in the vicinity of the microsome-bound arylsulfatase C. This was smaller than the over-all value for microsomes (-1.08 +/- 0.04 X 10(-3) elementary charge/A2; Masamoto, K. (1982) J. Biochem. 92, 365-371). This suggests that the anion concentration in the vicinity of the enzyme on microsomes is lower than that in the bulk aqueous phase and is higher than the average value at the microsomal surface when the salt concentration is low.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of xantham gum to suspend large particles during flow cytometric analysis and sorting

In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity approximately 9-fold. For suspensions of multicellular spheroids 100-400 microns in diameter the measured sedimentation velocity was approximately 9 times slower than that in medium alone. Thus, spheroids of 100 microns diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.     

Release of enzymes from quiescent fibroblasts during ATP depletion

The association between ATP depletion and enzyme release from cells has been described in two different ways: as a more or less linear dependence, or with a threshold value below which the enzyme release will start. We have investigated the association between ATP depletion caused by various metabolic inhibitors and enzyme release on quiescent fibroblasts. We found that the enzyme release never started before the ATP had decreased to a critical low level. Addition of glucose to cells while ATP was still above this critical level led to a regeneration of ATP and enzyme release did not occur. If ATP was lowered to 35-40% and kept there for 24 h, the enzyme release was minimal. These results support the threshold theory for release of enzymes from cells.     

Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

Simultaneous extraction of proteins and DNA by an enzymatic treatment of the cell wall of <Emphasis Type="Italic">Palmaria palmata</Emphasis> (Rhodophyta)

The extraction of proteins and DNA from seaweed tissue is difficult due to the presence of cell wall anionic polysaccharides that, after cell disruption, remain in the extraction medium as hydrocolloidal compounds. These compounds increase medium viscosity, thus limiting access to and, consequently, quantification of the soluble macromolecules such as proteins or DNA. This study describes a protocol that enables the simultaneous isolation of proteins and DNA from the red seaweed Palmaria palmata. It is based upon a specific form of hydrolysis using a mixture of cellulase and xylanase at different concentrations. This approach was carried out on samples collected during April and July, seasons in which differences in protein content have previously been reported. Our results confirm this report and also show that protein yield depends on the enzyme concentration used. As for DNA, this enzymatic digestion results in a much higher yield compared with the control. However, no seasonal differences are found for DNA and there is no clear link between the increase in DNA yield and the enzyme mixture concentration.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding

We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.     

Viscosity-dependent structural fluctuations in enzyme catalysis.

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.     

Effect of Endogenous and Exogenous Urate on the Uricase Formation by Streptomyces sp.

Cells of a strain of Streptomyces sp. were incubated with an equivalent quantity of urate, xanthine, 6,8-dihydroxypurine or hypoxanthine in a medium deprived of other nitrogen source. The amount of uricase produced by these cells was shown to differ significantly, increasing in the following order of purine bases added to the medium: urate, xanthine, 6,8-dihydroxypurine and hypoxanthine. Of these was only urate indicated to be the inducer of uricase formation, and the difference in the quantity of uricase produced was found to be based on the duration of enzyme formation. The rate of uricase formation was essentially identical regardless of the purine bases supplied to cells.

Allantoin was accumulated in medium in remarkably different manners depending on the purine bases, which suggested the diversity in the mode of generation of urate in cells. Urate was generated at the slowest rate in the cells incubated with hypoxanthine, although the largest amount of uricase was produced, However, urate supplied to cells at the same rate but from medium failed to support the enzyme formation when the activity increased to a certain level. In order that the same amount of uricase was produced by the cells incubated with the different purine bases, the initial concentration of the purine bases should be raised so that they could remain in medium for the same incubation time.

Intracellular compartmentalization that might segregate endogenous and exogenous urate and might cause the difference in “effeciency” of these urate molecules as the inducer of uricase formation has been discussed.     

Diffusional barrier in the unfolding of a small protein

To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.     

Effect of pH on coenzyme binding to liver alcohol dehydrogenase.

1. The transient-state kinetics of ligand-displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on-velocity and off-velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 10. 2. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogues to the enzyme . NAD+ complex. 3. Critical experiments have been performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.     

Modeling binding kinetics at the Q(A) site in bacterial reaction centers

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.