Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate in frozen model systems

The alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC-lactose in a temperature range of -28 to 20 degrees C. In the maltodextrin and CMC-lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC-lactose systems with low initial solute concentration, as a consequence of freeze-concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the "rubbery" state of the CMC and CMC-lactose systems. The WLF equation with system-dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Kinetics of Inhibition of Green Crab (Scylla Serrata) Alkaline Phosphatase by Sodium (2,2′-bipyridine) Oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2′-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by vanadate

Green crab (Scylla serrata) alkaline phosphatase is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by vanadate has been studied. The time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the enzyme in the presence of different Na3VO4 concentrations showed that, at each Na3VO4 concentration, the rate decreased with increasing time until a straight line was approached, the slopes of the straight lines being the same for all concentrations. The results suggest that the inhibition of the enzyme by Na3VO4 is a slow, reversible reaction with fractional residual activity. The microscopic rate constants were determined for the reaction of the inhibitor with the enzyme. As compared with Na2HPO4 (Ki = 0.95 mM), Na2HAsO4 (Ki = 1.10 mM), and Na2WO4 (Ki = 1.55 mM), the results suggest that Na3VO4 (Ki = 0.135 mM) is a considerably more potent inhibitor than other inhibitors.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by sodium (2,2'-bipyridine) oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

Modeling the kinetics of the pectin methylesterase catalyzed de-esterfication of pectin in frozen systems

The applicability of the William, Landel, and Ferry (WLF) equation with a modification to take into account the effect of melt-dilution and an empirical log-logistic equation were evaluated to model the kinetics of diffusion-controlled reactions in frozen systems. Kinetic data for the pectin methylesterase catalyzed hydrolysis of pectin in four model systems with different glass transition temperatures: sucrose, maltodextrin (DE = 16.5-19.5), carboxymethylcellulose (CMC) and fructose in a temperature range of -24 to 0 degrees C were used. The modified WLF equation was evaluated with a concentration-dependent glass transition temperature (T(g)) as well as the glass transition temperature of the maximally freeze-concentrated matrix (T(g)') as reference temperatures. The equation with temperature-dependent T(g) described the reaction kinetics reasonably well in all the model systems studied. However the kinetics was better described by a linear relationship between log(V(0)/V(0ref)) and (T - T(ref)) in all cases except CMC. The log-logistic equation also described the kinetics reasonably well. The effect of melt-dilution on reactant concentration was found to be minimal in all cases.     

Radial diffusion in gel for micro determination of enzymes. I. Muramidase, alpha-amylase, DNase 1, RNase A, acid phosphatase, and alkaline phosphatase

The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference.     

Enzymatic Activity versus Structural Dynamics: The Case of Acetylcholinesterase Tetramer

The function of many proteins, such as enzymes, is modulated by structural fluctuations. This is especially the case in gated diffusion-controlled reactions (where the rates of the initial diffusional encounter and of structural fluctuations determine the overall rate of the reaction) and in oligomeric proteins (where function often requires a coordinated movement of individual subunits). A classic example of a diffusion-controlled biological reaction catalyzed by an oligomeric enzyme is the hydrolysis of synaptic acetylcholine (ACh) by tetrameric acetylcholinesterase (AChEt). Despite decades of efforts, the extent to which enzymatic efficiency of AChEt (or any other enzyme) is modulated by flexibility is not fully determined. This article attempts to determine the correlation between the dynamics of AChEt and the rate of reaction between AChEt and ACh. We employed equilibrium and nonequilibrium electro-diffusion models to compute rate coefficients for an ensemble of structures generated by molecular dynamics simulation. We found that, for the static initial model, the average reaction rate per active site is ∼22-30% slower in the tetramer than in the monomer. However, this effect of tetramerization is modulated by the intersubunit motions in the tetramer such that a complex interplay of steric and electrostatic effects either guides or blocks the substrate into or from each of the four active sites. As a result, the rate per active site calculated for some of the tetramer structures is only ∼15% smaller than the rate in the monomer. We conclude that structural dynamics minimizes the adverse effect of tetramerization, allowing the enzyme to maintain similar enzymatic efficiency in different oligomerization states.     

Kinetic properties of enzyme populations in vivo: alkaline phosphatase of the Escherichia coli periplasm.

Studies were conducted to determine the role that diffusion may play in the in vivo kinetics of the Escherichia coli periplasmic enzyme, alkaline phosphatase (AP, encoded by the gene pho A). Passive diffusion of solutes, from solution into the periplasm, is thought to occur mainly through porins in the outer membrane. The outer membrane therefore serves as a diffusion barrier separating a population of periplasmic enzymes from bulk substrate. E. coli strains containing a plasmid with the pho A gene linked to the lac promoter were used in this study in order to vary the amount of enzyme per cell. Alkaline phosphatase assays were conducted with intact cells, and the substrate concentration at half-maximum velocity (normally the Km for the enzyme) was determined as a function of enzyme concentration per cell. The results showed that diffusion of substrate to the enzyme caused as much as a 1000-fold change in this parameter, compared to that of purified enzyme. This suggested that diffusion was the rate-limiting step of the enzymatic reaction in these cells. In agreement with this type of reaction, Eadie-Hofstee and Lineweaver-Burk plots were not linear. At their extremes, these plots represented two types of kinetics. At high substrate concentration, equilibrium of substrate between bulk solution and the periplasm was achieved, and the kinetic properties conformed to Michaelis-Menten. At low substrate concentrations, there were a large number of free (unbound) enzymes, and each substrate molecule that entered the periplasm, through the diffusion barrier, resulted in product formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of effective diffusion coefficient of substrate in gel particles with immobilized biocatalyst

A method of determining of the effective diffusion coefficient of substrate in a particle, where the diffusion and consumption of substrate by biocatalytic reaction are present simultaneously, was designed and experimentally verified. The method is based on measuring the overall rate of heterogeneous biocatalytic reaction in particles of varying diameter. The effective diffusion coefficient, D_e, was determined by fitting the measured reaction rates with the solution of the reaction-diffusion equation. The method is tailored for cases where the enzyme reaction is governed by the Michaelis-Menten kinetics. The value of K_m required for the solution of the mathematical model was adopted from the measurement of the kinetics of free cells, whereas the rate parameter, k₂, was optimized together with D_e. As an experimental model, the sucrose hydrolysis catalyzed by Ca-alginate-entrapped yeast cells was examined. The particle diameter varied in the range of 1.2–3.9 mm and the initial reaction rates were measured in a batch-stirred reactor at a sucrose concentration of 100 m . The D_e of sucrose at 30°C was found to be 2.9 · 10⁻¹⁰ m²s⁻¹.     

The association reaction of yeast alcohol dehydrogenase with coenzyme is partly diffusion-controlled in solvents of increased viscosity

The steady-state kinetics of the yeast and liver alcohol dehydrogenase catalyzed reduction of aldehydes were examined in solvent mixtures of increased viscosity. This was done to investigate the effects of diffusion control on the fast association of NADH with the enzymes. Both glycerol and sucrose were unsatisfactory as viscosogens, as they inhibited the enzyme, but poly(ethylene glycol)/water mixtures were satisfactory. The 5-fold faster reaction of yeast alcohol dehydrogenase with NADH is partly diffusion controlled, whereas the slower liver alcohol dehydrogenase reaction showed no diffusion effects. These results are consistent with a yeast alcohol dehydrogenase active site that has relatively little steric hindrance to NADH binding. It is estimated that contributions to this association reaction from diffusion control and chemical activation control are equal at a solvent viscosity of 10 cP. Thus, under physiological conditions of increased viscocity the NADH association may be significantly affected by diffusion effects. In order to estimate accurately the maximum diffusion-controlled rate constant from diffusion theory, the diffusion coefficients of NADH were measured in poly(ethylene glycol)/water mixtures and were found to vary inversely as the solvent viscosity raised to the power of 0.5. The non-Stokesian behaviour of molecules as large as NADH in polymer/water mixtures may be a serious limitation to the routine use of poly(ethylene glycol) as a viscosogen for diffusion studies.     

Steady-state enzyme kinetics in the Escherichia coli periplasm: a model of a whole cell biocatalyst.

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the limiting step for the enzyme in the periplasm was substrate (para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts.     

Enzyme immobilized in a packed-bed reactor: kinetic parameters and mass transfer effects.

To describe axial dispersion, particle film mass transfer, intraparticle diffusion, and the chemical reaction of the substrate for enzymes immobilized in porous particles in packed columns, we have developed mathematical models for first- and zero-order limits of Michaelis-Menten kinetics. Steady-state solutions were derived for both long and short column boundary conditions and for plug flow. Theory was compared to experiments by hydrolysis of sucrose catalyzed by invertase bound to porous glass particles. Steady-state conversions were measured for a range of flow rates. Pulse response experiments with inert packing were used to determine values of bed void fraction and particle porosity.     

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium

Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.     

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Chicken Intestinal Alkaline Phosphatase : I. The kinetics and thermodynamics of reversible inactivation II. Reactivation by zinc ions

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.     

Kinetic studies on dextransucrase from the cariogenic oral bacterium Streptococcus mutans

The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.     

Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.     

A collagen-alkaline phosphatase conjugate membrane: enzymatic kinetics in-vitro stability

Collagen-alkaline phosphatase membranes have been prepared, and their enzymatic kinetics and in-vitro stability analyzed. Collagen-alkaline phosphatase dispersions were prepared by complexation in aqueous alkaline solution and cast into membranes by controlled dehydration. These membranes were then crosslinked in glutaraldehyde solution, washed thoroughly, and dried. Crosslinking in glutaraldehyde confers increased stability of catalytic activity to these collagen-enzyme membranes, especially when compared to uncrosslinked collagen-alkaline phosphatase membranes assayed in a similar fashion. Crosslinking in glutaraldehyde also appears to inhibit gross leaching of the soluble enzyme from the carrier matrix. Apparent intrinsic kinetic properties of the collagen-alkaline phosphatase conjugate were analyzed in membranes of various thickness in order to determine the effect of internal diffusion resistances on the kinetics of the immobilized enzyme. The apparent Michaelis constant of the immobilized enzyme decreased as a function of decreasing membrane thickness, reaching an observed apparent Michaelis constant of 1.6mM at a membrane thickness of 0.2 mm. Extrapolation of the apparent Michaelis constant to zero membrane thickness, using a linear plot of the natural logarithm of the apparent Michaelis constant versus membrane thickness, allowed estimation of the true Michaelis constant of the immobilized enzyme. The estimated value for the true Michaelis constant of the collagen-alkaline phosphatase complex was 0.7mM. This value agrees closely with reported values for several purified mammalian alkaline phosphatase. The apparent Michaelis constant for the 0.2mm collagen-enzyme membrane agrees closely with the Michaelis constant reported for an alkaline phosphate purified from chondrocyte matrix vesicles. The intrinsic maximum reaction velocity (V(m)) of the collagen-enzyme complex was estimated b plotting the observed reaction rate as a function of decreasing membrane thickness and extrapolating such plots, at various substrate concentrations, to the limiting case of zero membrane thickness. The maximum reaction velocity was obtained by the common intercept of these plots as they approached zero membrane thickness.