Ionic control of biochemical reactions

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PEP-I-P-

FRUCTOSE

ious pts components in the cell is shown in Fig. 4. It stresses the direct interaction of Enzyme I with adenylate cyclase, a complex which directly affects the enzyme activity (see Fig. 3). The nature of the multienzyme complex indicates that the inhibition of adenylate cyclase by glucose demands the function of all the pts components. This working model explains the interaction of the pts system with adenylate cyclase and demonstrates that transport of sugars into the cell is coupled to inhibition of adenylate cyclase. References 1 Pastan, I. and Perlman, R. (1970) Science 169, 339-344 2 Lis, J.T. and Schleif, R. (1973) J. Mol. Biol. 79, 149162 3 Makman, R.S. and Sutherland, E. W. (1965) J. Biol. Chem. 240, 1309-1314 4 Peterkofsky, A., Harwood, J.P. and Gazdar, C. (1975) J. Cvclic Nucleotide Res. 1. 1l-20 Peterkofsky, A. and Gazdar, C. (1975) Proc. Nat. Acad. Sci. U.S.A. 72,292&2924

OUTSIDE

Peterkofsky, A. and Gazdar, C. (1974) Proc. Nat.

Fig. 4. The interaction of the pts svstem with adenylate cyclase. The model suggests that membrane-bound adenylate cyclase functions as a multi-enzyme complex regulated by the pts system. It is hypothesized that the interaction of adenylate cyclase with the Enzyme I protein is direct. The inhibition of adenylate cyclase activity coupled to sugar transport is represented to be indirect and dependent on the presence of all the components of the transport system.

II, A) [9]; however, the adenylate cyclase can no longer be inhibited by glucose. It was therefore concluded that, while Enzyme II is not required for the activity of adenylate cyclase, it is necessary for the regulatory effect of glucose. Studies on E. coli strains carrying leaky mutations in the general cytoplasmic proteins, Enzyme I or HPr (Table II, B) produced results of a different nature [5]. While a mutation in the HPr protein did not diminish the adenylate cyclase activity, a leaky mutation in the Enzyme I protein led to essentially complete loss in enzyme activity. The depressed levels of adenylate cyclase in the Enzyme I mutant were not the result of the absence of adenylate cyclase protein, but rather the ramification of a regulation defect; the addition of phosphoenolpyruvate fully restored the adenylate cyclase activity in the leaky Enzyme I mutant. It was concluded from these studies that Enzyme I plays an essential role in regulating adenylate cyclase activity [5].

low. The high activity state is favored by the presence of phosphoenolpyruvate and the absence of glucose, while the low activity state is favored by the presence of glucose and the absence of phosphoenolpyruvate. A comprehensive model indicating the location of adenylate cyclase and the var-

A phosphorylation-dephosphorylation model

It has been clearly established by experiments with the common bacterium Escherichia coli that it is possible for a cell to control its output of a given product in two ways: it may change the number of enzyme molecules available for some biochemical step in any sequential process

A current hypothesis to explain the interaction of ’the pts system with adenylate cyclase involves the idea that adenylate cyclase is normally complexed with Enzyme I (Fig. 3) [5]. When Enzyme I exists in a phosphorylated form, adenylate cyclase can express a high level of activity; when Enzyme I is dephosphorylated, adenylate cyclase activity is

T

Acad. Sci. U.S.A. 71,2324-2328

Harwood, J. P. and Peterkofsky, A. (1975) J. Biol. Chem. 250,4656-4662

Roseman, S. (1972) in The Molecular Basis of Biological Transport (Woessner, Jr, J.F. and Huijing, F., eds), pp. 181-215, Academic Press, New York Harwood, J.P., Gazdar, C., Prasad, C., Peterkofsky, A., Curtis, S.J. and Epstein, W. (1976) J. Biol. Chem. 251,2462-2468 Experiments in Molecular Genetics (1972) (Miller, J.H., ed.), pp. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor Peterkofsky, A. and Gazdar, C. (1971) Proc. Nat. Acad. Sci. U.S.A. 68,2794-2798

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lomc control of biochemical reactions Pierre Douzou and Patrick Maurel Ionic strength jluctuations play an essential role in the efficiency of enzyme-catalyzed reactions occurring inpolyelectrolyte microenvironmentsprovidedby the ‘biological organization’ and by the polyelectrolyte nature of some proteins and nucleic acids. This ionic control might play a major physiological role in regulating systems involved in genetic translation as well as in the cellular metabolism ofplant tissues and higher animals.

P.D. and P.M. are at the French Institut National de la Sante et de la Recherche Medicale. P.D. is also working at the Foundation Edmond de Rothschild, in Paris.

[ 1,2], or it may change their rate of reaction by inhibition or by activation, the inhibitors and activators usually being small molecules, to which the enzymes automatically respond by structural modifications [3,4]. These might not represent the sole mechanisms for regulating enzyme reactions in eukaryotic and prokaryotic cells, in both of which physiologically important control might also be exerted by ionic exchanges at the macromolecular-compo-

15

TIBS - January 1977 nent level, for systems involved in genetic translation as well as biomembranes. How could a change of cation levels within the cell and organelle compartments help in regulating biochemical reactions? We have tried to answer this question by studying enzyme-catalyzed reactions dependent on both pH and ionic strength. It has been known for decades that the pH optimum of an enzymatic reaction is not fully defined without specification of the ionic species present and its concentration, because in a large number of cases the pH optimum of activity is dependent on the ionic strength of the medium. However, the relationship established between enzyme activity and both pH and ionic strength has not provided a complete understanding of the process. This might be due partly to the fact that most kinetic studies elucidated only the overall activity and not the kinetic parameters k,,, and KmCappj, reactions usually being followed at an arbitrary substrate concentration sometimes lower than the KmCappj. Furthermore, these reactions were always analyzed as classical enzymic processes, specifically stimulated or inhibited by the ions, without taking into account the possible influence of the physical properties of the microenvironment on the catalysis. As we shall see later, most of the reactions which are pH and ionic-strength dependent are those in which enzymes interact with polyelectrolytic macromolecular cell components. These include systems involved in genetic translation as well as membrane-bound enzymes. Although the complexity of the cell cytoskeleton and membrane means that little is known about their electrical structure, there is increasing evidence that they are predominantly negatively charged. This is the case for the cell walls of bacteria like E. coli and Micrococcus luteus [5-81, for the surface of the inner membrane of mitochondria [9], and presumably for any membrane system containing sialoglycoproteins or sialoglycolipids [lo] and phospholipids such as cardiolipin, with a double-negative charge, found in mitochondrial membranes [ 111. These many tissue components are polyanionic and their character can be revealed by the strong fixation of polycations such as polylysine [5,7,8], ferritin [12] and by the effects of heparin and spermidin empirically used in the investigation of many biochemical reactions. Since enzymes are often themselves polyelectrolytes, specific catalytic implications can be expected from their interaction with polyanionic microenvironments. Thus, it seems necessary to undertake a reappraisal of both concept and experiment applied to enzyme systems

I

1

I

I

6

7

8

9

P

Fig. 1. Activity of membrane bound cytochrome c ox& dare (inner mitochondrial membrane) as a function of pH, at various ionic strengths: O-0, 0.01 M (KU); .-a, 0.06; A-A, 0.11; ? -_O, ? 0.21. Cytochrome c concentration, 10 PM. (Redrawn from ref. 19.)

functioning in the immediate vicinity of polyelectrolytic compounds, that is, of many membrane-bound enzymes and of enzymes involved in genetic translation. Ionic strength and enzyme kinetics A large number of enzyme systems presents a singular and not thoroughly understood behavior as a function of both pH and ionic strength. This is the case for example with the enzyme cytochrome c oxidase, extracted by Wainio et al. [13], from the inner membrane of mitochondria using the deoxycholate method and containing a large amount of phospholipids. This is again the case for Clostridium perfringens sialidase investigated with mixed sialoglycolipids as substrates by Barton et al. [lo], and for the lytic activity of lysozyme towards M. luteus cell walls [8,14]. Similar observations have been reported for the hydrolysis of RNA by ribonuclease [15,16], as well as for reactions involving small molecules as substrates [ 15,171. These results prompted us to undertake’ a detailed kinetic study of selected enzyme systems postulated as occurring in polyelectrolyte micro-environments, to verify this assumption, and finally to interpret-their behavior in terms of the

catalytic implications of electrostatic potentials, according to the well known polyelectrolyte theory [18]. A detailed kinetic study (k,,,, KmCappj) of the lytic activity of lysozyme on oligosaccharides (small substrates) and M. luteus cell walls was carried out as a function of both pH and ionic strength [8], with the following results. Whereas the hydrolytic activity of lysozyme on oligosaccharides has a pH optimum of 5.2 independent of ionic strength, the activity with the bacterial substrate was detected at very alkaline pH (optimum at pH 9.0) at low ionic strength (10.01 M), and the k,,, vs. pH proliles were shifted towards more acidic values as Zwas increased, approaching the one obtained for the hydrolysis of polysaccharides for Z=O.16 M. Moreover, as ionic strength increased, the lytic reaction appeared to be first activated then inhibited, this process being closely dependent on pH. The Michaelis constant (KmCappJ)was also strongly influenced by ionic strength. From systematic kinetic studies at normal and sub-zero temperatures in the presence and absence of synthetic polyelectrolytes (polycations and polyanions), we concluded that the cell walls possess a strong polyanionic character influencing the enzymic behavior of lysozyme [8]. A recent paper from Hackenbrock [ 121, showing that most of the cytochrome c oxidase of the inner mitochondrial membrane was located in a polyanionic environment, prompted us to investigate the activity of this enzyme towards added cytochrome c in relation to both pH and ionic strength [19], according to the kinetic pattern defined.for the lysozyme study. Some of our results on this system are shown in Fig. 1, in which it is seen that pH vs. activity profiles were shifted towards acidic values as Z was increased from 0.01 to 0.21. Further investigations [ 191 confirmed these results, which are consistent with earlier data from the literature [13], and clearly indicate that the lower the concentration of exogenous cytochrome c used as substrate, the greater the magnitude of the pH-profile shift. This is a result of great significance from a physiological point of view, the in vivo ratio cytochrome c oxidase/cytochromes c + ci being 1.5 and corresponding to a very low cytochrome c concentration. Catalysis in polyeleetrolyte environments The results obtained [8,19] were compared with those obtained by Goldstein et al. [18,20] on the catalytic behavior of trypsin and chymotrypsin covalently bound to insoluble polyelectrolytic carriers, and explained in terms of the polyelectrolyte theory [ 18,2 I]. A detailed

16

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H*\: H+

rrl

H+

r

-u

H+E

“+

H+ H+

w Fig. 2. Idealised representations of the various classes of enzyme systems functioning under the influence of electrostatic potentials. Class I: enzymes embedded in a microenvironment of macroscopic carriers (membranes). An example of a polyanionic microenvironment. Class II: enzymes acting on polyelectrolytic substrates (RNA, DNA etc.). Class III: enzymes themselves providing the electrostatic potential (either strongly acidic or basic proteins). An example of a very basic protein. Class IV: oligomeric enzymes in which a ‘catalytic’ subunit is under the influence of ‘regulatory’ subunits providing the electrostatic potential.

description of this theory and of its remarkable agreement with our experimental results has already been given by us elsewhere [8] and only a brief account sufficient to understand its main implications for catalytic behavior will be repeated here. Any polyanionic environment promotes a strong local negative electrostatic potential which acts on the physico-chemical properties of the medium and the enzyme. In particular the local proton concentration, termed (HL), is different from the bulk concentration, termed (Hz”,) according to the relation: pHi,=pHou,+0.43

e’f’/kT

where E is the unit charge and Y the local electrostatic potential promoted by the polyanion. Since Y is negative, pHi” is lower than pH,,,. In the presence of cations, this potential is modified by the electrostatic screening of the mobile charges, and according to the polyelectrolytic theory Y is a decreasing linear function of the logarithm of ionic strength. This relation, which was clearly verified

through the activity of the enzyme systems considered above, shows that pH and ionic strength are tightly interdependent within the polyanionic microenvironments. At low ionic strength, the pH profiles of such reactions, obtained by plotting activity against pHout, are therefore shifted towards alkaline values with respect to the normal conditions, that is in the absence of any electrostatic potential. Such shifts could also be explained, at least in some systems, by assuming a corresponding variation of the ionisation constant of the catalytic groups of the active site. Finally, as ionic strength increases, Y decreases, eventually vanishing and pHi” tends towards pHout, restoring the ‘normal’ conditions and therefore a pH optimum close to that found for the solubilized free enzymes. Membrane-bound enzymes According to a recent report [22] and in agreement with the mosaic model proposed by Singer [23], biological membranes present a heterogeneous structure. Domains presumably exist, within which certain membrane components predomi-

- Januar_v

1977

nate. The ionic nature of these components (phospholipids, sialolipids, sialoglycoproteins etc . . . .) implies that electrical heterogeneity accompanies the structural variation. Such electrical heterogeneity means that zones exist on the membrane, where high electrostatic potentials are generated. We have seen in the preceding sections of this review, as well as in ref. 8, how these potentials can control enzyme activity, and how they, can be modulated by ionic screening (Class I systems in Fig. 2). Genetic translation systems Proteins involved in genetic translation systems act under the influence of nucleic acids and therefore of strongly negative polyelectrolytes. (Class II systems, Fig. 2). On the other hand, some of the proteins interacting with these polyanions are very basic in character and might themselves behave as polycations, suffering and promoting modifications of the local electrostatic potentials which are in turn influenced by any ionic strength and (or) pH fluctuation. To test these assumptions it is worth considering one of the simplest systems available, that is ribonuclease A, performing a familiar hydrolysis reaction, the ribonuclease-catalyzed hydrolysis of cytidine 2’,3’-monophosphate [ 151shows a pH shift towards alkaline values when ionic strength is increased, as a consequence of the ribonuclease’s polycationic character (Class III systems, Fig. 2). However, the pH vs. activity profiles of the hydrolysis of the RNA (polyanion) by ribonuclease are displaced by about two pH units towards acidic values as ionic strength increases from 0.1 to 0.4 M [15,16]. Thus, in the first case the positive electrostatic field due to the ribonuclease influences the hydrolysis of the single nucleotide, but in the second case the strong negative field surrounding the RNA molecule influences the catalytic behavior of the ribonuclease. On the other hand, we found with this system [24] that the higher the pH of the medium (pH,,J, the lower the ionic concentration at which the activity optimum occurs. This is in agreement with the relationship pHi,=pH,“,+0.43 2YLkT. Working at constant pH,,, values, that is in conditions of any buffered internal environment, and modifying initially the local electrostatic potentials by addition of selected concentrations of polyamines (spermine, spermidine), we obtained a progressive shift of the bell-shaped activity curves towards lower NaCl concentrations, a result showing how variations in ‘local’ pH vaues, induced by these polyamines (polycations), influence the pH-dependent ribonuclease activity. Such patterns and interpretations could

17

TIBS - January 1977 be generalized to the sequential processes of genetic translation. These, studied at constant pH,.show characteristic bell-shaped curves as either ionic strength or ‘effector’ concentration is varied. Such results reveal the influence of these parameters on the pH dependence of the sequential processes, by modifying both the ‘local pH’ and the pK values of the ionizing groups concerned. Up to now very few experiments have been designed to reveal these regulating mechanisms. However, it has been shown [25] that the rates of alkaline hydrolysis of some esters of transfer RNA were influenced by ionic strength in such a way that the effect could be quantitatively explained on the basis of a polyelectrolyte effect due to the RNA molecule. More recently [26], it has been found that ribosoma1 subunits obey the polyelectrolyte theory, involving specific implications on the. -electrical properties of some ribosoma1 proteins. Component interactions and main reaction steps in the major stages of protein synthesis should be ‘revisited’ in the light of these preliminary results on vacant ribosomes, with the experimental and conceptual approach used for ribonuclease activity. The situation is then certainly much more complex than in the simplest systems investigated as ‘models’, partly because the structure of the nucleic acid moieties can be influenced, in particular through changes in the pK values of nucleic acid bases, and because a number of protein components could behave as polyelectrolytes providing electrostatic potentials and acting possibly as ‘regulatory’ systems. Allosteric enzymes

In a number of cases, complex enzyme behavior associated with allosteric regulations, could be explained by the polyelectrolytic nature of the enzyme, as well as by the presence of strongly charged substrates and effecters, able themselves to modulate the local electrostatic potential (Class IV systems, Fig. 2). Acidic and basic enzymatic proteins acting as polyanions or polycations, as well as ‘regulatory’ protein subunits able to exist in at least two distinct, electrically different forms, could undergo structural changes through electrostatic effects due to their own modulable electrostatic potentials. These mechanisms are under close examination in this Laboratory. Physiological control

The fact that proteins of a polyelectrolyte nature, and (or) provided with a polyelectrolytic microenvironment, are

specifically influenced by changes in ionic strength may well be of physiological importance, since experimentally induced changes are not usually very far from the possible fluctuations that may arise in vivo. Under these conditions, the physiological changes in proton and cation levels within the cell compartments could be understood through their ability to regulate metabolic processes by modulating the electrostatic potentials under which most macromolecular components of the cell machinery operate. This ‘physiological’ control would take place, together with the controls already established, in systems involved in genetic translation as well as in cells of plants and higher animals.

10 Barton, N., Lipovac, V. and Rosenberg, A. (1975) J. Biol. Chem. 250,8462-8466

11 Guamieri, M., Stechmiller, B. and Lehninger, A. L. (1971) J. Biol. Chem. 246, 7526-7532 12 Hackenbrock, C.R. (1975) Arch. Biochem. Biophys. 170, 139-148 13 Wainio, W. W., Eichel, B. and Gould, A. (1960) J. Biol. Chem. 235, 1521-1525 14 Davies, R.C. and Neuberger, A. (1969) Biochim. Biophys. Acta 178, 306317 15 Irie, M. (1965) J. Biochem. Tokyo. 57,355-362

16 Kalnitzky, G., Hummel, J.P. and Dierks, C. (1959) J. Biol. Chem. 234, 1512-1516 17 Sluyterman, L. A. A. and Degraaf, M. J. M. (1972) Biochim. Biophys. Acta 258,554-561

18 Goldstein, L., Levin, Y. and Katchalsky, E. (1964) Biochemistry 3, 1913-1919 19 Maurel, P. and Douzou, P. (1976) C. R. Acad. Sci. Paris 282,2107-2110

20 Goldstein, L. (1972) Biochemistry II, 40724084 21 Engasser, J. M. and Harvath, C. (1975) Biochem. J. 145,431-435

References 1 Monod, J. and Jacob, F. (1961) J. Mol. Biol. 3, 318-356 2 Jacob, F., Brenner, S. and Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, 329-348 3 Monod, J., Changeux, J.P. and Jacob, F. (1963) J. Mol. Biol. 6, 306-329 4 Monod, J., Wyman, J. and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118

5 Katchalsky, E., Bichowsky-Slonimsky, L. and Volcani, B.E. (1953) Biochem. J. 55,671680 6 Salton, M.R. J. (1964) Proc. 3rd Int. Symp. Fleming’s Lysosyme, Milan, p5/RT 7 Sela, M. and Steiner, L. A. (1963) Biochemistry

22 Lenaz, G., Landi, L., Cabrini, L. and Pasquali, P. (1975) Arch. Biochem. Biophys. 167, 744-753 23 Singer, S.J. (1972) Ann. N.Y. Acad. Sci. 195, 1623

24 Maurel, P. and Douzou, P. (1976) N. V Acad. Sci., submitted 25 Schuber, F. and Pinck, M. (1974) Biochimie 56, 397403

26 Kliber, S., Hui Bon Hoa, G., Douzou, P., Graffe, M. and Manago, M. Nucleic Acid Res., in press

2,41&421 8 Maurel, P. and Douzou, P. (1976) J. Mol. Biol.

102,253-264 9 Bosmann, B. H., Myers, M. W., Dehon, D., Ball, R. andCase, K.R.(1972) J. CellBiol. 55; 147-160

Cell fusion Jack A. Lucy The fusion of one somatic cell with another, which can lead to the production of interesting and biochemically useful hybrid cells, appears to have come of age. Chemicallykduced cell fusion, supported by an increased understanding of the membrane changes involved, may replace the fusion of cells by Sendai virus and lead to new uses of the technique. What is ‘cell fusion’? Anyone who is not familiar with this term might think that it is simply a synonym for fertilization. Certainly fertilization is a prime example of cell fusion, but fertilization also has some important special features. Firstly, germ cells contain only half the number of chromosomes that are present in somatic cells; this ensures, of course, that the number of chromosomes in somatic cells is constant from generation to generation. Secondly, the fusion of sperm and egg cells occurs with a high spontaneous frequency. Also, special mechanisms exist to ensure that multiple fertilization of an egg by more than one sperm cell does not norJ.A.L. is Professor of Biochemistry at the Royal Free Hospital School of Medicine, University of London, 8 Hunter Street, London WC1 N 1 BP, U.K.

mally occur. Finally, cross fertilization between the germ cells of differing species is possible only in a limited number of instances. ‘Cell fusion’ as commonly understood in the laboratory is, in some respects, the antithesis of fertilization since it involves the fusion of somatic cells. The homokaryons or heterokaryons produced therefore contain a full chromosome complement from each cell participating in the fusion process. By contrast to fertilization, somatic cell fusion is usually a rare spontaneous event. Until recently the routine use of cell fusion as a laboratory tool was thus feasible only by treating cells with inactivated Sendai virus to increase the frequency of fusion [ 1,2]. Many cells carry receptors for Send& virus. This makes it possible to