Transport catalysis

Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This “induced transition fit” (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for “low affinity” transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.     

Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis

Carrier-linked transport through biomembranes is treated under the view of catalysis. As in enzymes, substrate-protein interaction yields catalytic energy in overcoming the activation barrier. At variance with enzymes, catalytic energy is concentrated on structural changes of the carrier rather than on the substrate destabilization for facilitating the global protein rearrangements during transport. A transition state is invoked in which the binding site assumes the best fit to the substrate, whereas in the two ground (internal and external) states, the fit is poor. The maximum binding energy released in the transition state provides catalytic energy to enable the large carrier protein transformations associated with transport. This "induced transition fit" (ITF) of carrier catalysis provides a framework of rules, concerning specificity, unidirectional versus exchange type transport, directing inhibitors to the ground state instead of the transition state, and excluding simultaneous chemical and transport catalysis (vectorial group translocation). The possible role of external energy sources (ATP and Deltapsi) in supplementing the catalytic energy is elucidated. The analysis of the structure-function relationship based on new carrier structures may be challenged to account for the workings of the ITF.     

Transport viewed as a catalytic process

Transport catalysis is analysed in terms of the "induced transition fit" (ITF) concept. The essentials of ITF are briefly elucidated, emphasizing the difference of substrate-protein interactions between enzymes and carriers exemplified by the paradigm ADP/ATP carrier (AAC). Two of the numerous applications of the ITF are discussed in more detail: unidirectional passive and active transport and the relation of substrate site type inhibitors to the carrier conformations. According to ITF in most cases of unidirectional transport intrinsic binding energies may be insufficient for transport catalysis and requires additional energy from ATP or electrochemical gradients. The impacts of external energy on the carrier cycle are examined for ABC transporters (mdr) and for cation-substrate co-transporters (LacY). The relations of inhibitors to the binding site of the carrier are discussed, given the paradigm examples of side specific inhibitors of the AAC. Results with the AAC suggest the induction of an abortive ground state by inhibitors, representing extreme side specific conformation of the binding center.     

Role of substrate binding forces in exchange-only transport systems: I. Transition-state theory

Summary An analysis of transition-state models for exchange-only transport shows that substrate binding forces, carrier conformational changes, and coupled substrate flow are interrelated. For a system to catalyze exchange but not net transport, addition of the substrate must convert the carrier from an immobile to a mobile form. The reduction in the energy barrier to movement is necessarily paid for out of the intrinsic binding energy between the substrate and the transport site, and is dependent on the formation of two different types of complex: a loose complex initially and a tight complex in the transition state in carrier movement. Hence the site should at first be incompletely organized for optimal binding but, following a conformational change, complementary to the substrate structure in the transition state. The conformational change, which may involve the whole protein, would be induced by cooperative interactions between the substrate and several groups within the site, involving a chelate effect. The tightness of coupling, i.e., the ratio of exchange to net transport, is directly proportional to the increased binding energy in the transition state, a relationship which allows the virtual substrate dissociation constant in the transition state to be calculated from experimental rate and half-saturation constants. Because the transition state is present in minute amount, strong bonding here does not enhance the substrate's affinity, and specificity may, therefore, be expressed in maximum exchange rates alone. However, where substrates largely convert the carrier to a transport intermediate whose mobility is the same with all substrates, specificity is also expressed in affinity. Hence the expression of substrate specificity provides evidence on the translocation mechanism.     

A high affinity fungal nitrate carrier with two transport mechanisms

We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H(+)-coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H(+), and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two Michaelis-Menten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes.

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitiors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces , is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

Design principles for active transport systems

We have previously described simple models for active transport and have derived steady state equations for the unidirectional flux of substrate in terms of a minimal set of kinetic parameters. Here we consider how to maximize the pumping rate of a carrier-enzyme through the optimal utilization of the ATP hydrolysis reaction. The equations for net flux contain rate constants and dissociation constants and these determine the maximum velocities and affinities measured in transport kinetic analysis. It is assumed that the rate constants can evolve to the diffusion limited rate of substrate binding as has apparently occurred in the enzyme triosephosphate isomerase (Knowles & Albery, 1977). The dissociation constants of the rate limiting intermediates fit the affinities for substrates on different sides of the membrane and are dependent on the basic free energy levels (Hill, 1976) of the carrier substrate system. From our analysis it is clear that there are three ways to design a system with optimal affinities and that the choice is linked to the sequence of substrate binding. It is possible to use free energy differences of isomerization (Boyer, 1975) or ligand-ligand interactions (Weber, 1975) both of which have been described previously, but which are incorporated here into a unified treatment. A third possibility is to couple the binding step of a transported ligand to the progress of a chemical reaction as might occur, for example, if Na⁺ must be bound before the carrier can be phosphorylated. In this way the free energy of hydrolysis can be used not only to drive the overall pumping reaction, but also to fix differentially the affinity for substrate on either side of the membrane, as required for rapid pumping.     

Are there both low-and high-affinity glutamate transporters in rat cortical synaptosomes?

Kinetics of sodium dependent glutamic acid transport have been studied in rat cortical synaptosomes at sufficiently high glutamic acid concentrations ([G]) to delineate the low affinity transporter. Computer optimization techniques were used to fit the data to models which account for the sodium and substrate dependence of uptake. The data fit about equally well models consisting of two carriers (Model 1) or one carrier plus a linear component (Model 2). However, the results of further studies were inconsistent with Model 1, but totally consistent with Model 2. Thus the results are incompatible with the presence of both high-and low-affinity carriers. The carrier model found in previous studies of high affinity glutamate transport predicts the effects of high [G] and [Na] observed in the present study. The biphasic effect of [Na] on velocity of uptake is the logical consequence of the operation of this model. The rate equation for this model has been utilized to define and compute kinetic parameters which characterize the transporter. These kinetic functions are remarkably similar in shape and magnitude to previous estimates from the studies of the high affinity transport (low [G]). The results of other studies by the author which corroborate and expand the predictions of the kinetic model are discussed. These have been combined with the present results to formulate a rather comprehensive model of glutamate function. This model can be used to describe function in terms of mathematical equations and to make predictions from these equations. These equations relate velocity of uptake and the kinetic parameters to sodium and substrate concentration, velocity to membrane potential, distribution ratio to the electrochemical potential, and release to time, compartment sizes, and exchange constants. Such processes as concentration in the presynaptic terminal, depolarization induced release, re-uptake following stimulus induced release, and postsynaptic depolarization are all possible consequences of the operation of this model. The wide applicability of the model to the transport of other substrates in addition to glutamate is discussed.     

Expression of substrate specificity in facilitated transport systems

Summary In facilitated transport systems the carrier reorientation step is shown to be largely independent of the forces of interaction between the substrate and the carrier site, whereas in coupled systems (obligatory exchange or cotransport) reorientation proceeds at the expense of the binding force developed in the transition state. In consequence, the expression of substrate specificity is expected to differ in the two systems. In the facilitated transport of analogs no larger than the normal substrate, the affinity but not the maximum rate of transport can vary widely; with larger analogs, both the affinity and rale can vary if steric constraints are more severe in the translocation step than in binding. In coupled transport, by contrast, the translocation step can be highly sensitive to the structure of the substrate, and binding much less sensitive. The theory agrees with published observations on facilitated systems for choline and glucose in erythrocytes, as well as on Na⁺-coupled systems for the same substrates in other cells. The following mechanism, which could account for the behavior, is proposed. In facilitated systems, the transport site fits the substrate closely and retains its shape as the carrier undergoes reorientation. In coupled systems, the site is initially looser, but during carrier reorientation it contracts around the substrate. In both systems, the carrier encloses the substrate during the translocation step, though for a different reason: in coupled but not in facilitated systems the binding force enormously increases in the enclosed state, through a chelation effect. In both systems, steric interference with enclosure retards the translocation of bulky substrate analogs.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Facilitated diffusion with consecutive reaction: optimal carrier affinity

The interplay between facilitated diffusion of a substrate through a membrane and a consecutive enzymic reaction, both of which follow Michaelis-Menten kinetics, has been theoretically investigated and the effect of the kinetic and transport parameters on the rate of substrate uptake is graphically illustrated. At steady state two characteristic features of the system have been identified. First, the substrate concentration at the internal enzymic side of the membrane cannot exceed a given value even at much higher external substrate concentrations. Second, the uptake rate is maximum at a given value of K_T, the kinetic parameter of the transport system that expresses the reciprocal carrier affinity of the substrate. The optimum value of K_T is approximately equal to the external substrate concentration. This particular dependence of the uptake rate on the carrier affinity is expected to play an important role in hormonal regulation.     

Reaction mechanism of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Kr?mer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.     

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.     

Effects on transport of rapidly penetrating,competing substrates: Activation and inhibition of the choline carrier in erythrocytes by imidazole

Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.     

Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate. Probes for the energy barrier to uncoupled transport

The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.     

Kinetic Evidence for the Uniport Mechanism Hypothesis in the Mitochondrial Tricarboxylate Transport System

The kinetics of the transport of citrate by the tricarboxylate transport system located in the inner mitochondrial membrane was studied in proteoliposomes containing the purified carrier protein, in order to verify the previously hypothesized mechanism of uniport (J. Bioenerg. Biomembr. 35, 133–140, 2003) and achieve some information on the kinetic properties of the carrier transport system. For this purpose, a mathematical model has been elaborated and the experimental data were analyzed according to it. The results indicate that the data actually fit with the uniport model, and hence it is confirmed that the carrier has a single binding site for its substrates and can oscillate between the inside and outside form, in both the free and substrate-bound states. The rearrangement of the free form is slower than the bound form in both directions. The dissociation constants for the internal substrate are at least one order of magnitude higher than the one for external citrate. As a consequence of these last two points, the rate of citrate transport by the carrier is much higher when it operates in exchange with another substrate than when it operates in net uniport.     

The choline transport system of erythrocytes. Distribution of the free carrier in the membrane

A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.