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R M Krupka

Publications and source records attributed to R M Krupka.

At least 19 recordsLinked to original sources
PubMed1999

Uncoupled active transport mechanisms accounting for low selectivity in multidrug carriers: P-glycoprotein and SMR antiporters.

The extraordinarily low substrate specificity of P-glycoprotein conflicts with the notion that specific substrate interactions are required in the control of the reaction path in an active transport system. The difficulty is shown to be overcome by a half-coupled mechanism in which the ATP reaction is linked to carrier transformations, as in a fully coupled system, but in which the transported substrate plays a passive role. The mechanism, which requires no specific interaction with the substrate, brings about uphill transport. A half-coupled mechanism is directly supported by two observations: (i) almost completely uncoupled ATPase activity in purified P-glycoprotein, and (ii) a pattern of substrate specificity like that of passive systems, where maximum rates for different substrates vary little (unlike active systems, where maximum rates vary greatly). The mechanism accommodates other findings: partial inhibition of ATPase activity by an actively transported substrate; simultaneous binding and translocation of more than one substrate molecule; and stimulation or inhibition of the transport of one substrate molecule by another. A half-coupled system associated with an internal competitive inhibitor should behave as if tightly coupled, in agreement with the effects of the synthetic peptide, polytryptophan. The degree of coupling in the intact system is yet to be determined, however. A half-coupled ATPase mechanism could originally have evolved in a flippase, where immersion of the carrier in its substrate, the membrane lipid, precludes uncoupled ATP hydrolysis. These concepts may have wider application. An uncoupled antiport mechanism, driven by a proton gradient rather than ATP, can explain low selectivity in the SMR multidrug carriers of bacteria, and a half-coupled mechanism for the ion-driven cotransport of water (the substrate in which the carrier site is immersed) can explain a recently proposed uphill flow of water.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Limits on the tightness of coupling in active transport.

Control of the coupled reaction sequence in active transport depends on systematic changes in the properties of the carrier protein as the reaction proceeds. These changes would have to be brought about by specific interactions with the substrate, the binding forces being used to stabilize either (i) a carrier state with altered properties or (ii) the transition state in a carrier transformation. In the first case the tightness of coupling (the ratio of the coupled rate to slippage) will at first rise with the increment in binding energy in the altered state but will approach an upper limit when overly strong binding forces retard substrate dissociation in a subsequent step in the coupled reaction sequence. Primary and secondary active transport are subject to this limitation because the coupling mechanism necessarily involves intermediates in which the substrate is strongly bound. Exchange-only transport is not necessarily subject to the same limitation because the mechanism can involve only a substrate-catalyzed change in carrier state. The available data, although scant, agree with these conclusions.

Biological Transport, Active↗
PubMed1998

Channelling free energy into work in biological processes.

The process by which free energy from ATP or an ion gradient is coupled to work--either osmotic work, by a membrane pump, or mechanical work, by a molecular motor--as well as the development of force by a molecular motor, can be explained by mechanisms dependent on substrate binding energy. Coupling involves a reaction sequence that combines the driving and driven reactions and that is controlled at switch points where the mobility of the coupling protein and its specificity in binding and catalysis can be abruptly altered; the altered state is an intermediate (or transition state) in the coupled reaction, the unaltered state an intermediate in the uncoupled reaction (slippage). The balance between these states, which is determined by the increase in substrate binding energy in the transformation, decides the relative importance of the coupled and uncoupled paths. On this basis a general expression for switching may be derived: the tightness of coupling is limited by a ratio of substrate dissociation constants before and after a controlling change in state: Rate(coupled)/Rate(uncoupled) < or = Kinitial state/Kfinal state. When binding energy is used in this way to distort the conformation of a protein, mechanical work is done; the work is internal but becomes external if the protein is connected to an external load. External work, force F multiplied by distance d, is then limited by a ratio of substrate dissociation constants before and after the conformational change: W = Fd < RT In(Kinitial state/Kfinal state). The ratios of dissociation constants, estimated from published values of the force exerted by molecular motors, are > 1 x 10(4) for myosin and > 2 x 10(4) for kinesin. From the tightness of coupling of the anion exchange carrier of red cells the ratio of constants for coupling is > or = 4 x 10(4). These increments can be accounted for by conversion of a surface complex to an enclosed complex, as in chelate formation. The work done by an ATP-driven motor is limited by the free energy of ATP hydrolysis, but the force developed is independent of the ATP reaction, and comes from binding energy with either the driving or driven substrate.

Adenosine Triphosphate↗
PubMed1996

Force generation, work, and coupling in molecular motors.

A mechanism is proposed for molecular motors in which force is generated by a protein conformational change driven by binding energy (in muscle, that of myosin with actin as well as with ATP, ADP, or Pi). Work, the product of the force generated by one myosin or kinesin molecule (F) and the distance over which it acts (d), is a function of a ratio of dissociation constants before and after the contractile step: F.d < RT ln(KAe/KAc). From published data the ratio is > 2 x 10(4), which can be explained by conversion of a surface complex to an enclosed, or partly enclosed, complex. Although the complex performing the work stroke is in unstrained conformation, the complex after the work stroke is much more stable, owing to binding forces; the latter, however, is destabilized by the load, which thereby opposes the contractile conformational change, countering the force-generating reaction. The connection between the free energy release and work is implicit in the mechanism, inasmuch as coupling, like force generation, depends on conformational changes driven by binding energy (internal rather than external work being involved in coupling). The principles apply whether ATP or an ion gradient drives the system. At high load, in muscle, the mechanism allows for a summation of the forces generated by several myosin molecules.

Actins↗
PubMed1995

Interpreting the effects of specific protein modification on antiport coupling mechanisms: the case of the aspartate/glutamate exchanger.

Reaction of two cysteine residues in the aspartate/glutamate carrier of mitochondria is reported to abolish exchange but to actuate a passive one-way exit of extremely low substrate affinity and specificity, but with the same activation energy as antiport (Dierks, T., Salentin, A. and Krämer, R. (1990) Biochim. Biophys. Acta 1028, 281-288). This behaviour, reminiscent of a channel, becomes understandable when the required control over carrier mobility by the substrate is allowed for. Whether the transport mechanism involves a substrate site alternately exposed on opposite sides of the membrane or sites simultaneously exposed on both sides, and whether the substrate acts by converting an immobile carrier conformation to an inherently mobile intermediate or by stabilizing the transition state in carrier movement, the same fundamental relationship emerges: the ratio of coupled to uncoupled rates (antiport relative to net flux) is limited by the ratio of substrate dissociation constants in successive carrier conformations, one immobile, the other mobile; the increment in the binding energy in the two forms must therefore be large. Shifts in the equilibrium between these conformations and shifts in their relative affinities for the substrate can account for the properties of the modified transport system, which, it is concluded, functions as a carrier, not a channel.

Antiporters↗
PubMed1994

Interpreting the effects of site-directed mutagenesis on active transport systems.

Single amino acid substitutions in the lactose permease of Escherichia coli are known to elicit behaviour, such as the transformation of an active into a passive system, not explained by current co-transport models. The behaviour, it is shown, can be explained by an expanded reaction scheme that takes account of the required alternation of the carrier, in the course of the coupled reaction, between mobile and immobile conformations or between conformations that bind either only one substrate or both substrates. The extended model links such behaviour to altered conformational equilibria or binding regions. Thus, mutations that affect the equilibrium between a mobile one-site conformation of the free carrier and an immobile conformation having sites for both substrates allow passive transport of the second substrate in an ordered mechanism, and mutations in a secondary substrate binding region that affects this conformational change allow passive transport of the first substrate. Mutations in regions interacting with a substrate in the transition state in carrier movement, as well as in the initial binding sites, can also be distinguished. The analysis applies to both primary and secondary active transport.

Biological Transport, Active↗
PubMed1994

The application of vectorial coupling theory to the calcium pump.

Models for the calcium pump of the E1E2 type appear to be inconsistent with new evidence for the binding of internal and external calcium ions, simultaneously, to the free pump. The models are shown here to be incomplete but not necessarily wrong; they omit the required modulation of the mobility, specificity, and enzyme activity of the pump, which is brought about through substrate-controlled conformational changes. A reaction scheme based on the E1E2 model but incorporating the conformational equilibria in question is shown to account for a variety of experimental findings, including those at odds with the simple model: (i) binding of lumenal Ca2+ to the free as well as the phosphorylated pump; (ii) uncoupled exit of lumenal Ca2+ at high concentrations; (iii) the absence of any effect of lumenal Ca2+ on the binding of external Ca2+; (iv) uncoupled ATPase activity in dimethyl sulfoxide.

Biological Transport, Active↗
PubMed1993

Coupling mechanisms in active transport.

In primary and secondary active transport, the mobility and specificity of the carrier are controlled, over the course of the transport reaction, in accordance with a set of 'rules'. The rules are shown to depend on two mechanisms: a substrate--either the driving substrate (a transported ion or ATP) or the driven substrate--may shift a conformational equilibrium or accelerate a rate-limiting conformational change. From an analysis of coupling mechanisms the following conclusions emerge. (i) The ratio of coupled to uncoupled flux, which should be large, cannot be greater than the ratio of substrate dissociation constants in an initial complex and a conformationally altered state. A minimum value for the increased binding force can be estimated from steady-state constants. (ii) In an ordered mechanism, slippage is expected at high concentrations of the substrate adding to the carrier second, while slippage of the first substrate should remain low. (iii) Slippage in coupled transport is minimized if the driven substrate is last on in loading the carrier and last off in unloading, while the reverse order makes the affinity high in loading and low in unloading, as required for efficient transfer from one compartment to another; hence the preferred mechanism may depend on prevailing physiological conditions. (iv) A coupled transport system can be transformed into a facilitated system for one substrate or both if the control of carrier mobility is undermined through modification of the carrier.

Biological Transport, Active↗
PubMed1993

Coupling mechanisms in ATP-driven pumps.

Because the kinetic reaction schemes for primary and secondary active transport can be identical, the same fundamental relationship holds among rate and equilibrium constants: the ratio of coupled to uncoupled flux is no greater than the ratio of substrate dissociation constants in an initial complex and a conformationally altered state. Further, the role played by each substrate in coupling depends in the same way on its order of addition to the carrier. It follows that the structural principles governing the design and operation of the carrier proteins are fundamentally alike. In either system, the strict control of the mobility and specificity of the carrier, a prerequisite for active transport, depends on the utilization of substrate binding forces to alter the protein conformation; and whether the driving substrate is transported or not and whether reversibly bound or covalently bound (like the phosphate group derived from ATP), the force producing the conformational change is derived from non-covalent interactions between the substrate (held at the substrate site) and other sections of the protein. The protein probably encloses the substrate, with a resulting increase in the binding force; the favourable energy of interaction balances the unfavourable energy involved in distorting the protein structure. The postulated complex can account for the 'occluded state' of transported cations and for the favourable reaction of inorganic phosphate with the calcium pump.

Adenosine Triphosphate↗
PubMed1992

Kinetics of transport systems dependent on periplasmic binding proteins.

Rate equations are derived for a transport model involving a water-soluble binding protein outside the plasma membrane. On addition of the substrate, the conformation of the binding protein changes; the complex then combines with the membrane carrier, transferring the substrate to the carrier site. The free binding protein leaves and the carrier shifts inward, releasing the substrate inside the cell. Exit follows the reverse path. The predicted behaviour is as follows. (i) Uptake does not necessarily conform to Michaelis-Menten kinetics. (ii) In both the energized and de-energized states, the maximum rate of exit is far lower than that of entry; the asymmetry is determined by the conformational change in the binding protein, which is independent of the energy state of the system. (iii) Exchange transport is inhibited by external substrate and is extremely slow; consequently counter-transport is not expected. (iv) The half-saturation constant in uptake can differ from the dissociation constant of the binding protein. (v) The maximum rate of uptake depends on the intrinsic substrate affinity of the membrane carrier relative to that of the binding protein. (vi) The maximum rate of uptake and the substrate half-saturation constant depend on the concentration of the binding protein.

Biological Transport↗
PubMed1992

Testing models for transport systems dependent on periplasmic binding proteins.

A carrier model in which transport across the cytoplasmic membrane is mediated by a periplasmic binding protein (Krupka, R.M. (1992) Biochim. Biophys. Acta 1110, 1-10) is shown to account for many of the properties of these systems: (i) Michaelis-Menten kinetics; (ii) seemingly irreversible uptake; (iii) the absence of exchange transport and counter-transport; (iv) substrate half-saturation constants that in different systems may be lower or higher than the dissociation constant of the binding protein; (v) the high concentration of the binding protein in the periplasm and its weak association with the membrane component. The binding protein appears to function as a valve or rectifier that permits the substrate to enter the cell, but blocks exit in both the energized and de-energized states. The asymmetry depends on both the abruptness and the extent of the conformational change in the binding protein. Characteristically, these systems build up steep gradients across the membrane, circumstances in which such a valve might be important. In agreement with the mechanism, (a) the binding protein is missing in members of the same family of transporters that function in export of the substrate rather than import; and (b) in Gram-positive organisms, which have no periplasmic space, binding proteins function while anchored to the cytoplasmic membrane.

Biological Transport↗
PubMed1990

A simple test for the sidedness of binding of transport inhibitors.

A new method is described for determining the sidedness of action of nonpolar inhibitors that rapidly diffuse through the lipid bilayer and could therefore interact with the carrier on both sides of the membrane. Sidedness is deduced from the effect of the inhibitor on the flux ratio for the substrate (the ratio of the rates of exchange and net transport). The advantages of the method are that the experimental measurements are made after the inhibitor has equilibrated rather than in the brief period when it is present on only one side of the membrane, and that any reversible inhibitor can be tested, whether the inhibition mechanism is competitive, noncompetitive, uncompetitive, or mixed.

Binding Sites↗
PubMed1990

Inhibition of choline transport in erythrocytes by n-alkanols.

The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.

1-Butanol↗
PubMed1990

Expression of substrate specificity in facilitated transport systems.

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 rate 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.

Biological Transport↗
PubMed1989

Testing transport models and transport data by means of kinetic rejection criteria.

In the case of a transport system obeying Michaelis-Menten kinetics, completely general relationships are shown to exist between the final ratio of internal and external substrate concentrations, alpha, and the V/Km ratios found in zero-trans-entry, zero-trans-exit and equilibrium-exchange experiments (where V is a maximum substrate flux and Km a substrate half-saturation constant). The proof depends on a new method of derivation proceeding from the form of the experimental data rather than, as has been the practice in kinetic analysis, from a hypothetical reaction scheme. These general relationships, which will be true of all mechanisms giving rise to a particular type of behaviour (here Michaelis-Menten kinetics), provide a test for internal consistency in a set of experimental data. Other relationships, which are specific, can be derived from individual reaction schemes, with the use of traditional procedures in kinetic analysis. The specific relationships include constants for infinite trans entry and exit in addition to constants involved in the general relationships. In conjunction, the general and specific relationships provide a stringent test of mechanism. A set of results that fails to satisfy the general relationships must be rejected; here systematic error or unexpected changes in the transport system in different experiments may have distorted the calculated constants, or the system may not actually obey Michaelis-Menten kinetics. Results in accord with the general relationships, on the other hand, can be applied in specific tests of mechanism. The usefulness of the theorem is illustrated in the cases of the glucose-transport and choline-transport systems of erythrocytes. Experimental results taken from several studies in the literature, which were in accord with hyperbolic substrate kinetics, had previously been shown to disagree with relationships derived for the carrier model, and the model was rejected. The new analysis shows that the data violated the general relationships and therefore cannot decide the issue. More recent results on the glucose-transport system satisfy the general relations and agree with the carrier model.

Biological Transport↗
PubMed1989

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

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.

Carrier Proteins↗
PubMed1989

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells.

The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate subsites are required for the preferred substrates, Cl- and HCO3-, to account for tight binding in the transition state (Kdiss congruent to 1 microM). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unusual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and "tunneling," which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.

Anion Transport Proteins↗