ECC 1989 INDEX...

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01. Electroconformational Coupling 1989
02. ABSTRACT
03. Introduction
04. Cyclic Process of Enzyme Catalysis
05. Electroconformational Coupling
06. Activation of membrane ATPASE by oscillating electric fields
07. Mitochondrial ATPASE from beef heart
08. Interpretation the ECC model
09. Relation to other cellular energy and signal transductions
10. ACKNOWLEDGEMENTS



01. Electroconformational Coupling 1989
p. 319 Bioelectrochemistry and Bioenergetics, 21 (1989)

319-331 A section of J. Electroanal. Chem., and constituting Vol. 275 (1989) Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

Electroconformational coupling (ECC): an electric field induced enzyme oscillation for cellular energy and signal transductions* Tian Yow Tsong, Dao-Sheng Liu and Francoise Chauvin Department of Biochemistry, University of Minnesota College of Biological Sciences, 1479 Gortner Avenue, St. Paul, MN 55108 (U.S.A.)

Aldolfas Gaigalas and R. Dean Astumian, National Institutes of Standards and Technology, Chemical Process Metrology Division, Gaithersburg, MD 20899 (U.S.A.)

(Received 20 November 1988; in revised form 4 February 1989)


02. ABSTRACT
Previous work has shown that membrane ATPases can extract free energy from applied oscillating electric fields for doing chemical work, e.g. to synthesize ATP from ADP and P(i) or to transport Rb and Na ions against their respective electrochemical gradient. Data of these experiments are briefly reviewed. Electroconformational Coupling (ECC) is used to interpret these results. Computer analysis of a four state cyclic enzyme mechanism reproduces many experimental features. It is shown that a coulombic interaction between an enzyme and an alternating electric field (ac) can cause the enzyme to oscillate between different conformational states. If the frequency of the applied field matches the kinetic characteristics of the system and the amplitude matches the energy required for inducing productive catalytic cycling, a phenomenological resonance between catalytic reaction and the periodic field is generated. A condition necessary for achieving energy coupling is the kinetic bias arising from the binding energy of the ligand. Analysis indicates that only dynamic electric fields, i.e. oscillating or fluctuating fields, can propel the cyclic reaction of the enzyme catalysis, and thus be effective for transducing energy. A stationary transmembrane electric field must be modulated, e.g. by opening and closing of an ion channel, to become oscillatory in order to produce the same effect. We propose that ECC is a fundamental process of cellular energy and signal transductions. Here, many membrane associated events are reduced to Michaelis-Menten types of enzyme catalytic reactions and they are thus amenable to the quantitative analysis of chemical kinetics.


03. Introduction
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Electrochemical potential of ions have been postulated to play a major role in free energy transductions and information transfer of cells. In neural transmission, Na and K currents are responsible for the generation and propagation of the action potential [1,2]. In mitochondrial ATP synthesis, the proton gradient across the inner membrane is the high energy intermediate, which, upon translocation of protons along the electrochemical gradient, transfers its potential energy to ATPase for the synthesis of ATP [3-8]. In photosynthetic processes, the energy of a photon is used to pump a proton into an energy reservoir and ATP synthase then uses the electrochemical potential energy of the proton for synthesis of ATP [7,9,10]. Notwithstanding, there is no compelling evidence which would exclude a direct energy transfer between the electric field and a protein, thus allowing a temporary storage of energy in the conformational states of the protein [11]. Previously, we proposed a mechanism, Electroconformational Coupling (ECC), to test the feasibility of direct energy transaction between a transmembrane electric field and an enzyme conformational equilibrium for driving ion pumps and ATP synthesis [12-15]. Here we will summarize new experimental evidence and analysis based on the concept of ECC. We will examine and compare the ECC model and the common enzyme catalytic process as exemplified by the Michaelis-Menten Mechanism.

The electric potential across cell membranes is of the order of 10 to 250 mV, which corresponds to a field intensity of 20 to 500 kV/cm. Under such a strong field, molecules will behave quite differently than they will under the zero field condition. Ion pairs will dissociate, dipoles will orient, molecules will be electronically and automatically polarized, equilibria between different conformers of a protein will be shifted, etc. [16,17]. There are two geometric situations under which these changes can take place. The first is where all molecules and ions freely diffuse. The second is when they are fixed relative to the field direction. The first situation is represented by a reaction in an homogeneous aqueous solution. The field effect on the rapidly tumbling molecule is generally small and has been discussed elsewhere [12,16,17]. Here we will focus on the second situation which is more relevant for dealing with effects of an electric field on a membrane protein. Let us start by considering a general enzyme catalytic reaction.


04. Cyclic Process of Enzyme Catalysis
An enzyme catalytic process is a cyclic reaction because the enzyme is recycled at each turnover. A cyclic process will respond to a periodic driving force with which the enzyme can interact. As a result of this interaction, the enzyme will oscillate between its different conformational states. This phenomenon has been shown to have an implication in cellular membrane processes. To examine the cyclic behavior of an enzyme, we will consider the simple Michaelis-Menten mechanism (scheme 1 of Fig. 1). The enzyme bonds to the substrate to form an enzyme-substrate complex.

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(Fig. 1. Cyclic enzyme catalytic process. Many membrane processes mediated by receptors and enzymes exhibit kinetic characteristics similar to the (?-missing) of a Michaelis-Menten enzyme. Such an enzyme is susceptible to periodic perturbation. This paper considers how oscillating electric fields can interact with membrane ATPases and in so doing induce enzyme conformational oscillations, thus allowing utilization of the binding energy of ligands for catalyzing endergonic reactions. See text for details.)

The product is then released and the initial enzyme state is regenerated when the complex dissociates. The driving force of this reaction is the negative free energy of the S to P conversion. In fact, with a non-reversible step at the product releasing step, the reaction is implied to proceed to the left even if the free energy has a positive sign. Enzyme recycling has a specific rate, given by the turnover rate of the Michaelis-Menten mechanism. Generally, most investigators agree that there is another state preceding the formation of the product, namely the enzyme-product complex, as shown in scheme 2. If the two reversible steps are much faster than the dissociation of the enzyme- product complex, the kinetics of scheme 2 will be indistinguishable from that of scheme 1, and scheme 2 is in essence a Michaelis-Menten mechanism. In the third scheme, we simply rewrite the second scheme in a more consistent manner. It becomes a cyclic mechanism. Again, the reaction is driven by the negative free energy of the S to P conversion, although the description of the process is inherently unidirectional since it is shown to proceed only in the clockwise direction. To be more precise, the enzyme state which favors the binding substrate must be different from the state which favors the binding of the product state. A distinction between E(1) and E(2) is necessary. The Michaelis-Menten mechanism of scheme 1 is now more generally written as scheme 4. However, scheme 4 has an inconsistency. We all accept that without an additional (p. 322)


05. Electroconformational Coupling
One example of a periodic or oscillating driving force in a living cell is the transmembrane electric field. Although the transmembrane potential of a cell has been considered constant in the past, recent analyses suggest that locally it may exhibit large amplitude oscillations of fluctuations when time (is) resolved to ms or us, (microsecond), levels. Many membrane integral proteins have been shown to be electrically active. For examples, the VDAC (voltage-dependent anion channel) from the outer membrane of the mitochondria [18], the Na-channel/batrachotoxin complex [19], and the acetylcholine receptor [20] open in specific ranges of transmembrane electric potential and close in other ranges. These field dependent conformational changes may be coupled to ligand binding processes for energy and signal transductions [12-15]. Let us consider a simple two state conformational transition,

(formulae) (1)

The equilibrium constant of the reaction is K = k(1)/k(-1). The properties which make a protein responsive to an electric field are its electric moment (u) and polarizability (a). The molar electric moment of P(1) is M(1) = u(1) + a(1)E and of P(2) , M(2) = u(2) + a(2)E. The change in molar electric moment for the conformational transition is (delta)M = M(2) - M(1). An electric field of strength E will shift the equilibrium according to the generalized van't Hoff equation,


06. Activation of membrane ATPASE by oscillating electric fields
Experiments on the activation of [Na,K]-ATPase of human erythrocytes and mitochondrial F(0)F(1)ATPase of beef heart by oscillating electric fields are good examples of how membrane proteins can respond to oscillating electric fields, capture energy and couple that energy to drive endergonic reactions.

[Na,K]-ATPase of human erythrocytes

Previous experiments from this laboratory indicated that [Na,K]-ATPase of human erythrocytes might be susceptible to electric perturbation and might be one of the sites of electroporation when an electric field of the order of kV/cm is used to induce pores in human red cells [23]. A membrane conductance which could be blocked by ouabain was detected. We then attempted to active this enzyme using a field which would generate a transmembrane potential of the order of 10 mV. Human red cells in an isotonic suspension were exposed to applied alternating electric fields of strengths up to 50 V/cm (peak-to-peak) and of frequencies between 1 Hz and 20 MHz. The transport of Na+ and Rb+ (or K+) into and out of the cytoplasm was measured by radioactive tracers and compared with control samples in which no ac field was imposed. The activity which was inhibited by ouabain was taken to be due to the catalytic action of the [Na,K]-ATPase. Other inhibitors of the enzyme such as oligomycin, vanadate and oubagenin also inhibited the electric field provoked activity. In contrast, inhibitors to anion transport and Na+/Na+ exchange have no appreciable effects except at concentrations 10-1000-fold higher than that required to inhibit these activities. The results of these experiments are summarized below [24-27].

(1) At 4 (degrees) C, the maximum ac stimulated activity (i.e. at optimum field strength and optimum frequency) was 15-20 ions/pump for the Rb+ uptake and 20-30 ions/pump for the Na+ efflux. The ratio of Na+ pump activity and Rb+ pump activity was roughly 3/2, although it was not strictly maintained for any one red cell sample.


07. Mitochondrial ATPASE from beef heart
Witt and co-workers [28] were the first to report activation of ATP synthesis of chloroplasts by pulsed electric fields. The yield of ATP was less than one per enzyme per electric pulse and it was assumed that the electric field triggered the release of tightly bound nucleotides from the enzyme surface. Our experiments were performed with beef heart mitochondrial ATPase and our goal was to demonstrate that a single pulse of electric field can induce enzyme turnover [29-33]. The electron transport activity of submitchondrial particles was inhibited by rotenone or cyanide or both. Thus, the energy source for ATP synthesis, about 42 kJ/mol, must be derived from the applied electric field. Using the capacitor discharge technique it was shown that ATP was formed from ADP and P(i) and this synthesis was inhibited completely by oligomycin and FCCP (carbonyl cyanide p-triflouromethoxyphenol- hydrazone), the former being a potent inhibitor and the latter an ionophore of proton. When dithiothreitol was present in the medium, as much as 5 ATP molecules per enzyme were generated with a single pulse of 30 kV/cm (300 mV of transmembrane potential) with a decay constant of 100 us. We interpret these results as due to the oxidation-reduction cycle of certain SH groups in the ATP synthetic process. Firgure 5A shows some results of these experiments using the exponentially decaying electric field.

Alternating electric fields have also been used to demonstrate ATP synthesis. In most experiments, we used an electric field of 60 V/cm at various frequencies. The ATP yield per ac cycle is low (approximately 10^-4) because of the low energy level of the applied field. However, the experiment is simple and the results are essential for clarifying certain concepts of the ECC model, as discussed below.

Another interesting observation is that the natural inhibitor peptide of the ATPase has a profound effect on the electric field induced ATP synthesis. Synthesis was effective only if the peptide was removed. Figure 5B gives some of the results which show an increase in ATP yield as the natural inhibitor peptide dissociates with time from the enzyme.


08. Interpretation the ECC model
The main features of the above results on [Na,K]-ATPase have been predicted from computer analyses of the ECC model using eqn. (3) [12-17]. These include the ability of eqn. (3) to absorb energy from an applied field for actively pumping a neutral substrate or an ion, an optimum field strength and an optimum frequency for this effect. Computer analysis, shown in Fig. 4B, reproduces the frequency dependence of the Na+ pump and the Rb+ pump. No quantitative fit of the data

p. 329
points was attempted. The difference in the frequency for effective pumping for Na+ and Rb+ was produced by adjusting the number of gating charges (2 for Rb+ pump and 3 for Na+ pump) and the rate constants of eqn. (3). Details of the simulation will be presented elsewhere.

The results on the ATP synthesis experiment need further elaboration. According to the ECC model, only a dynamic electric field (either oscillating or fluctuating) can induce a phenomenological resonance with the enzyme cyclic reation and be effective for energy coupling. An exponentially decaying electric field can be viewed as a fourier sum of many components, each with a characteristic frequency. Among these components are high frequency ones which would be able to induce enzyme turnover within a single electric discharge. This is judged unlikely to be the reason for enzyme turnover as the amplitude of these higher frequency components is bound to be small and the interaction energy (delta M*E) would be insufficient for ATP synthesis.

The above consideration led us to suggest that within the mitochondrial ATPase a mechanism exists which can modulate a stationary transmembrane electric field to become locally oscillatory [12-17]. We suggested that the F(o) subunit could fill this role by translocating protons at a defined time interval. When a proton approaches from one side of the membrane and then translocates to the other side, the electric potential experienced by the enzyme would temporarily change its sign, making it oscillatory. There are many mechanisms for modulating a stationary electric potential. An ion channel, a redox protein, a charge motion, etc. in the vicinity of an energy transducing enzyme can effectively produce an oscillating field under an energy sustaining constant potential. We mentioned that oxidation and reduction of certain SH groups may be involved in the turnover of the mitochondrial ATPase. The mechanisms and details of its involvement remain to be investigated.


09. Relation to other cellular energy and signal transductions
We propose that the experiments and analyses presented above are reminiscent of how a cell transduces energy and signals [15,33,34]. A crucial question is whether the transmembrane potential of a cell is stationary or oscillatory. If it is oscillatory, the ECC model would be applicable for understanding the function of many membrane enzymes and receptors. If it is stationary, one would expect that such a potential be modulated by certain mechanisms before it can play a role in the cellular energy and signal transductions. Several methods of modulation may be conceived: (1) opening/closing of a channel protein, as in F(o)F(1)ATPase and the generation and propogation of an action potential, (2) altering the charge density of a protein, as in phosphorylation /dephosphorylation, (3) a redox reaction that transports electrons through a specified path, as in the electron transport chain, (4) binding/dissociation of ions or charged ligands, etc.

These ideas have been discussed previously. Most cellular events mediated by membrane receptors or enzymes basically have the character of a Michaelis- Menten enzyme, as depicted in scheme 5 of Fig. 1. Such a system is able to receive, decipher

p. 330
and respond to a periodic perturbation, i.e. an energy source or a signal, insofar as the receptor or enzyme is capable of interacting effectively with that periodic driving force. A reverse reaction will, in turn, allow the receptor or the enzyme to transmit energy or a signal. If the concept discussed here is near the truth, most useful information of the transmembrane electric activity of a cell is not contained in the measured stationary potential but rather in the local and time dependent fluctuations of the electric field.

Very weak electric fields (mV/cm) of various frequencies have been shown to trigger gene expression, stimulate RNA and protein biosynthesis, and influence cell differentiation, proliferation, bone healing, etc. (see e.g. refs. 35- 37). These phenomena may be understood using the concept of the ECC model, as well. Our task is then to identify cellular components that respond to these weak electric fields. In our previous work, we have examined how a very weak periodic field is amplified at the cite of the cell membrane [11,12,38,39]. With such mechanisms, organisms can communicate and navigate with extremely weak oscillatory electric, magnetic, acoustic or chemical potentials.


10. ACKNOWLEDGEMENTS
The work of T.Y.T., D.-S.L. AND F.C. has been supported by the United States Office of Naval Research and a National Science Foundation grant to T.Y.T. Contributions of our former and present associates and collaborators, Drs. K. Kinosita, E.H. Serpersu, J. Teissie, H.V. Westerhoff, P.B. Chock, Y.-D. Chen, B.E. Knox and F. Kamp, are greatly appreciated.

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