A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system.     

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.     

The role of water in the thermodynamics of dilute aqueous solutions

Water plays a role in the thermodynamics of dilute aqueous solutions that is unusual in two ways. First, knowledge of hydration equilibrium constants of species is not required in calculations of thermodynamic properties of biochemical reactants and reactions at specified pH. Second, since solvent provides an essentially infinite source of oxygen atoms in a reaction system where water is a reactant, oxygen atoms are not conserved in the reaction system in dilute aqueous solutions. This is related to the fact that H₂O is omitted in equilibrium expressions for dilute aqueous solutions. Calculations of the standard transformed Gibbs energies of formation of total carbon dioxide and total ammonia at specified pH are discussed, and the average bindings of hydrogen ions by these reactants are calculated by differentiation. Since both of these reactants are involved in the urease reaction, the apparent equilibrium constants and changes in the numbers of hydrogen ions bound are calculated for this reaction as functions of pH.     

Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1

In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.     

pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [14C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H+ greater than Cs+ greater than Rb+ greater than K greater than Na+ greater than Li+ greater than Mg2+, Ca2+. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondiffusible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Immobilized cycling enzyme active transport systems. pH feedback control

Active transport can be induced by applying a pH gradient across a membrane containing a homogeneous mixture of two cycling enzymes. When the two reactions are inversely 'pH active', one producing protons and the other consuming them, a pH feedback control of the functional structure occurs and the active transport function of the membrane can be either stabilized or inhibited according to whether the endogenic pH modifications tend to enhance or reduce the exergonic pH gradient. When it is stabilized, the system looks like a thin active layer surrounded by two diffusive layers, leading to a fairly good model for biological transport systems. Under particular conditions, signals can be emitted.     

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.     

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P.     

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).     

Ammonium/urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii.

The influence of ammonium and urea on the components of the proton electrochemical potential (delta p) and de novo synthesis of ATP was studied with Bacillus pasteurii ATCC 11859. In washed cells grown at high urea concentrations, a delta p of -56 +/- 29 mV, consisting of a membrane potential (delta psi) of -228 +/- 19 mV and of a transmembrane pH gradient (delta pH) equivalent to 172 +/- 38 mV, was measured. These cells contained only low amounts of potassium, and the addition of ammonium caused an immediate net decrease of both delta psi and delta pH, resulting in a net increase of delta p of about 49 mV and de novo synthesis of ATP. Addition of urea and its subsequent hydrolysis to ammonium by the cytosolic urease also caused an increase of delta p and ATP synthesis; a net initial increase of delta psi, accompanied by a slower decrease of delta pH in this case, was observed. Cells grown at low concentrations of urea contained high amounts of potassium and maintained a delta p of -113 +/- 26 mV, with a delta psi of -228 +/- 22 mV and a delta pH equivalent to 115 +/- 20 mV. Addition of ammonium to such cells resulted in the net decrease of delta psi and delta pH without a net increase in delta p or synthesis of ATP, whereas urea caused an increase of delta p and de novo synthesis of ATP, mainly because of a net increase of delta psi. The data reported in this work suggest that the ATP-generating system is coupled to urea hydrolysis via both an alkalinization of the cytoplasm by the ammonium generated in the urease reaction and a net increase of delta psi that is probably due to an efflux of ammonium ions. Furthermore, the findings of this study show that potassium ions are involved in the regulation of the intracellular pH and that ammonium ions may functionally replace potassium to a certain extent in reducing the membrane potential and alkalinizing the cytoplasm.     

Changes in binding of hydrogen ions in enzyme-catalyzed reactions

Most enzyme-catalyzed reactions produce or consume hydrogen ions, and this is expressed by the change in the binding of hydrogen ions in the biochemical reaction, as written in terms of reactants (sums of species). This property of a biochemical reaction is important because it determines the change in the apparent equilibrium constant K' with pH. This property is also important because it is the number of moles of hydrogen ions that can be produced by a biochemical reaction for passage through a membrane, or can be accepted from a transfer through a membrane. There are two ways to calculate the change in binding of hydrogen ions for an enzyme-catalyzed reaction. The first, which has been used for a long time, involves calculating the partial derivative of the standard transformed Gibbs energy of reaction with respect to pH. The second involves calculating the average numbers of hydrogen ions in each reactant and adding and subtracting these average numbers. The changes in binding of hydrogen ions calculated by the second method at pHs 5, 6, 7, 8, and 9 are given for 23 enzyme-catalyzed reactions. Values are given for 206 more reactions on the web. This database can be extended to include more reactions for which pKs of reactants are known or can be estimated.     

Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.     

Relationship between phosphorylation potential and electrochemical H+ gradient during glycolysis in Streptococcus lactis.

Assays of intracellular ATP, ADP, and inorganic phosphate allowed calculation of the phosphorylation potential (delta G'ATP/F) maintained during glycolysis by Streptococcus lactis. At the same time, the electrochemical H+ gradient (delta mu-H+/F) was evaluated by distribution methods, using radioactive tetraphenylphosphonium bromide as a probe for the membrane potential and salicylic acid as a probe for the pH gradient. Detailed comparisons were made at pH 5, when the reaction mediated by the proton-translocating ATPase (BF0F1) was likely to have been poised near equilibrium; for those conditions, the ratio delta G'ATP/delta mu-H+ was used to estimate stoichiometry for BF0F1 during ATP hydrolysis. At an external pH of 5, in the presence or absence of valinomycin, this ratio was close to 3, over a range of 370 to 510 mV (8.5 to 11.7 kcal/mol) for delta G'ATP/F and a range of 128 to 167 mV for delta mu-H+/F. Other work suggested that delta G'ATP/delta mu-H+ increased from its minimum value of 3 to 4.3 as the external pH changed from pH 5 to 7.     

Intramolecular proton-transfer reactions in a membrane-bound proton pump: the effect of pH on the peroxy to ferryl transition in cytochrome c oxidase

In the membrane-bound redox-driven proton pump cytochrome c oxidase, electron- and proton-transfer reactions must be coupled, which requires controlled modulation of the kinetic and/or thermodynamic properties of proton-transfer reactions through the membrane-spanning part of the protein. In this study we have investigated proton-transfer reactions through a pathway that is used for the transfer of both substrate and pumped protons in cytochrome c oxidase from Rhodobacter sphaeroides. Specifically, we focus on the formation of the so-called F intermediate, which is rate limited by an internal proton-transfer reaction from a possible branching point in the pathway, at a glutamic-acid residue (E(I-286)), to the binuclear center. We have also studied the reprotonation of E(I-286) from the bulk solution. Evaluation of the data in terms of a model presented in this work gives a rate of internal proton transfer from E(I-286) to the proton acceptor at the catalytic site of 1.1 x 10(4) s(-1). The apparent pK(a) of the donor (E(I-286)), determined from the pH dependence of the F-formation kinetics, was found to be 9.4, while the pK(a) of the proton acceptor at the catalytic site is likely to be > or = 2.5 pH units higher. In the pH range up to pH 10 the proton equilibrium between the bulk solution and E(I-286) was much faster than 10(4) s(-1), while in the pH range above pH 10 the proton uptake from solution is rate limiting for the overall reaction. The apparent second-order rate constant for proton transfer from the bulk solution to E(I-286) is >10(13) M(-1) s(-1), which indicates that the proton uptake is assisted by a local buffer consisting of protonatable residues at the protein surface.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Bioactive polymers. 56: urease immobilization on carboxymethylcellulose

This article reports on the reaction of urease immobilization through its covalent bonding on carboxymethylcellulose. The reaction is activated by dicyclohexylcarbodiimide. The coupling reaction is influenced by the enzyme-support and activator-support ratios, as well as by duration. Starting from a rotating, composed experimental program of the second order, the function correlating the activity of the immobilized enzyme with the reaction parameters is established. Immobilized urease exhibits thermal stability higher than that of free enzyme, regarding both pH and the inhibiting action of some metal ions or organic substances. The stability over time of the immobilized urease is high, its enzymatic activity being maintained at over 85% of the initial value three months after synthesis.     

Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes.

Ammonium ion and proton concentration profiles near the surface of a planar bilayer lipid membrane (BLM) generated by an ammonium ion gradient across the BLM are studied by means of microelectrodes. If the concentration of the weak base is small compared with the buffer capacity of the medium, the experimental results are well described by the standard physiological model in which the transmembrane transport is assumed to be limited by diffusion across unstirred layers (USLs) adjacent to the membrane at basic pH values (pH > pKa) and by the permeation across the membrane itself at acidic pH values. In a poorly buffered medium, however, these predictions are not fulfilled. A pH gradient that develops within the USL must be taken into account under these conditions. From the concentration distribution of ammonium ions recorded at both sides of the BLM, the membrane permeability for ammonia is determined for BLMs of different lipid composition (48 x 10(-3) cm/s in the case of diphytanoyl phosphatidylcholine). A theoretical model of weak electrolyte transport that is based on the knowledge of reaction and diffusion rates is found to describe well the experimental profiles under any conditions. The microelectrode technique can be applied for the study of the membrane permeability of other weak acids or bases, even if no microsensor for the substance under study is available, because with the help of the theoretical model the membrane permeability values can be estimated from pH profiles alone. The accuracy of such measurements is limited, however, because small changes in the equilibrium constants, diffusion coefficients, or concentrations used for computations create a systematic error.     

pH gradient across the lysosomal membrane generated by selective cation permeability and donnan equilibrium

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [¹⁴C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H⁺ ? Cs⁺ > Rb⁺ > K⁺ Na⁺ > Li⁺ ? Mg²⁺, Ca²⁺. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondifussible anions and (2) a selective permeability of the lysosomal membrane to cations.     

松嫩草甸3种主要植物群落土壤脲酶的初步研究

 松嫩草甸羊草（Leymus chinensis）群落、碱茅（Puccinellia tenuiflora）群落和虎尾草（Chloris virgata）群落土壤脲酶活性的季节动态呈单峰曲线变化,在土体中随土层的加深,其活性逐渐递减。各群落0～10 cm土层中的土壤脲酶活性与月平均降雨量呈幂函数关系,与土壤温度呈指数函数关系。土壤脲酶活性受多种土壤理化因子的共同影响,对于羊草群落,各因子的影响程度依次为：pH值>有机质>速效氮>C/N>容重>全氮>速效磷;碱茅群落为：有机质>C/N>全氮>容重>速效磷>速效氮>pH值;虎尾草群落为：全氮>有机质>速效氮>pH值>C/N>速效磷>容重。对该地区土壤肥力影响因子的主成分分析表明：有机质、全氮、速效氮、C/N和土壤容重对土壤肥力的贡献率占主导地位,土壤脲酶活性所占的比重较小,它不能完全反映土壤肥力状况。