Biochirnica et Biophysica Acta, 415 (1975) 149-171 ' 9 Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - Printed in The N e t h e r l a n d s BBA 85146

THE

REACTION

MECHANISM

OF THE

SODIUM

PUMP

R. W H I T T A M a n d A. R. C H I P P E R F I E L D

Department of Physiology, The University, Leicester LE 1 7RH (U.K.) (Received December 24th, 1974)

CONTENTS !.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Stepwise M e c h a n i s m

. . . . . . . . . . . . . . . . . . . . . . . . . .

B. Evidence Against the Stepwise 1. The Role o f K ~ . . . . . 2. The Binding of P~ . . . . . 3. O u a b a i n Binding . . . . . 4. The influence of pH . . .

Mechanism . . . . . . . . . . . . . . . . . . . . . . . .

C. The T ransition State M e c h a n i s m il.

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150

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152 152 153 154 155

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156

Kinetic Analysis of the Reaction Sequence . . . . . . . . . . . . . . . . . . . . .

157

A. The E n z y m e - S u b s t r a t e - M o d i f i e r Analysis . . . . . . . . . . . . . . . . . . . .

157

B. Kinetic Tests . . . . . . . . . . . . . 1. Sequences Involving A T P . . . . . . 2. The I n d e p e n d e n c e of the Na ÷ a n d K * 3. Evidence for the R a n d o m A t t a c h m e n t

. . . . . . . . . . . . . . . . . .

158 158 159 160

C. L i m i t a t i o n s and Scope of the Kinetic A p p r o a c h . . . . . . . . . . . . . . . . .

161

. . . . . . . . . . . . . . . . Binding Sites? . of Na ÷ a nd K ÷ .

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I ll. The Kinetic Basis for Affinity C h a n g e s . . . . . . . . . . . . . . . . . . . . . . A. Mutual Effects of K ÷ and A T P . I. The A T P a s e reaction . . . . 2. K ÷ as a Product I n h i b i t o r . . 3. The S p o n t a n e o u s T u r n o v e r of B. ITP and U T P Hydrolysis C. The Role of Mg 2÷

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . the P h o s p h o r y l a t e d I n t e r m e d i a t e . . . .

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162 163 163 163 164

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164

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164

IV. The Branched T r a n s i t i o n State M e c h a n i s m

. . . . . . . . . . . . . . . . . . . .

A. The Definition of the Branched M e c h a n i s m

. . . . . . . . . . . . . . . . . .

B. The Evidence for the Branched M e c h a n i s m . . . I. Internal K ÷- its Role in O u a b a i n Binding . . 2. Internal K ÷- its Role in Na÷: Na ÷ E x c h a n g e 3. Internal K ÷- its Role in K÷: K ÷ Exchange . 4. Flexibility in N a - : K ÷ C o u p l i n g . . . . . . 5. O u a b a i n and O l i g o m y c i n Sensitivities . . . .

V.

150

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165 165 167 167 167 167 167 167

C. The Physiological Role of Internal K + . . . . . . . . . . . . . . . . . . . . .

168

D. T h e Simplicity of the P u m p M e c h a n i s m . . . . . . . . . . . . . . . . . . . .

168

C o m p a r i s o n of the P u m p M e c h a n i s m with other Energy T r a n s d u c i n g Systems . . . . .

References

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169 170

150 I. INTRODUCTION Two cardinal questions about the sodium pump need to bc answered before its mechanism can be understood. The first relates to the chemical composition of the protein and phospholipid which comprise the pump in the membrane. The details of amino acid sequence and protein structure, the nature of the enzyme active site, the molecular weight, immunological properties and other aspects already make a substantial body of essential evidence. The role of phospholipid and especially its interaction with protein is likewise being clarified. The best work in this field is done with those preparations which approach purity in the sense that unwanted protein and lipid have been removed. Nevertheless, there is an inherent limi'ation to the insight that can be obtained with this approach. The very act of destroying the membrane to isolate the relevant components, inevitably restricts interpretation in terms of actual ion movements across the cell membrane. The aim, of course, is eventually to prepare a well-defined, man-made, intact membrane with vectorial properties in which ion movements associated with ATP hydrolysis can be measured. Until this breakthrough is made, work with natural membranes and intact cells is still needed to tackle the second central question which is whether Na ÷ and K ÷ are transported simultaneously or one after the other. Put another way: is the sodium pump, as regards ATP hydrolysis, a one-step or two-step reaction? We shall limit ourselves in this article to work bearing directly on this issue. Some justification is needed for a review on a single topic, whose fundamental significance may perhaps not be appreciated. The point concerns energy transduction and how the pump hydrolyses ATP such that Na + and K + are transported in opposite directions across the membrane. There is no ATP hydrolysis by the pump unless both Na + and K- are present. The reaction mechanism by which energy from ATP is coupled to the ion movements clearly depends on whether there is a one-step or two-step chemical reaction. Since the reaction is inextricably linked to the transport, the reactants are not just the chemicals whose bonds are changed but also those ions which are changed in location. Internal Na + and external K + are substrates, and external Na + and internal K + are products, Formulation of the reaction mechanism requires a decision whether there is simultaneous or separate transport of the two ions. This point is so central that we shall give an appraisal of the evidence pointing to separate transport, and then develop the arguments and present the evidence that lead us to the alternative view that the pump transports N a ' and K + simultaneously and h~drolyses ATP in a one-step reaction.

IA. The Stepwise Mechanism Experiments on squid axons and red cells showed clearly that there is an energy-dependent process which uses ATP, causing Na ÷ to be moved outwards and K + inwards so that high intracellular K + concentrations can be maintained in the face of high extracellular Na + concentrations and despite downhill leak fluxes of both ions [1~,]. When Skou found that in crab nerves there was an ATPase which required

151 both Na ÷ and K +, as well as Mg 2+, for optimal activity, it was recognised at once that this was probably the enzyme responsible for the active transport [5]. Proof that the (Na + -t- K+)-ATPase and the transport system were consubstantial came later [6-8] with the demonstration that the two phenomena shared several common features; the affinities for Na + and K + were comparable [6], both were specifically inhibited by cardiac glycosides [6,7] and both were similarly asymmetric [8]. Moreover, there is a correlation amongst different tissues between the amount of the enzyme and the rate of active transport [9]. In spite of a great deal of subsequent research [9-16] there has been very little experimental work on the question whether the pump is a one-step or two-step reaction. In essence, the pump can operate in only one of two possible ways. First, one ion may bind, be transported then be replaced by the other ion, which is carried back. Second, both ions may bind simultaneously and be transported in opposite directions in a single step. Although someyears ago, both mechanisms were considered as possibilities, a theoretical kinetic analysis did not resolve the question unambiguously [17], and measurements of ATP hydrolysis were not accurate enough to reach a conclusion [18]. In contrast, studies of phosphorylation reactions seem to be rather unambiguous and there is no doubt that much of this evidence is in keeping with a two-step reaction. The main point is that with Na + 4 - A T P + Mg 2÷ a phosphoprotein is formed, which is then hydrolysed in a reaction requiring K ÷ [19]. The suggestion is that Na + effiux is associated with the formation o f a phosphorylated intermediate and K + influx with its destruction at a later time [10,20]. A rather more elaborate mechanism with two phosphorylated intermediates instead of one was subsequently proposed and the essential features may be shown as follows [19,21]:

INSIDE NO+ ATP ADP

NO +

"Pi

/L OUTSI DE

NO

K"

! K*

K" (1)

"

This scheme has been almost universally accepted because it fits in well with other observations, of which one of the most important is the binding of cardiac glycosides. This work has great significance because the molecular configuration of the pump is indicated according to whether or not the glycoside is bound. The conditions for binding have attracted much attention, especially as the results were seemingly closely connected with the reaction mechanism depending on phosphoproteins. The chief results are that digoxin became bound predominantly in conditions where the phosphorylated intermediate was formed [22]: thus, it bound only if Na ÷ + A T P -- Mg 2÷ were present and not if K ÷ was also present. Later, two ouabain-binding pathways were identified [23], one being the sodium-dependent pathway already mentioned and the other was in the presence of Mg 2+ + Pt when

152 there was inhibition by Na ÷. A cyclic scheme was proposed to explain these results [I 3,24,25] : M g 2°, N o * AT

~"

* E I ~,

21 Mg

°

K" Pi

E2 ~

IEI P . A D P

I

Mg .

E2P*

~

H20

(2)

.-ouo bain

ouoboin

Pi* ouobain-

ouoboin-

enzyme

enzyme-

compl~'x

corn plex

P

There are three features of this scheme. First, is the assumption that the partial reactions do actually occur one after the other as separate and distinct steps in the main pathway for ATP hydrolysis and coupled ion movements as shown in the reaction scheme !. Second, P~ can become covalently bound to the enzyme and forms the same intermediate (E2P) as when the pump is phosphorylated by ATP in the presence of Na + plus Mg 2+. Third, ouabain binding can be entirely accounted for if it is confined to the putative E2 conformations. We shall now examine whether these three features provide a satisfactory basis for the reaction mechanism.

lB. Evidence Against the Stepwise Mechanism IB-I. The Role of K +. The stepwise mechanism is based primarily on the sensitivity to K ÷ and A D P of the phosphoprotein found with Na +, ATP and Mg 2+ [24]. It is the influence of Mg 2÷ that led to the view that there are two intermediates, El P and E2P; the observation was that at low Mg 2÷ concentrations the intermediate was not responsive to K + but undergoes transphosphorylation with A D P and takes part in an A T P - A D P exchange reaction. In contrast, at high Mg 2+ concentrations the intermediate was hydrolysed with K +, but it did not react with ADP. The effect of high Mg 2+ concentrations was abolished by N-ethylmaleimide and oligomycin. The transformation of EIP to E2P is held to be the step at which the selectivity changes from being high for Na + to being high for K ÷, but there is no direct evidence on this point. These results have had great impact regarding the mechanism and as they have been amply confirmed, there can be no question concerning their validity [9-16]. Moreover, it is undeniable that the partial reactions do lend support to the view that there is a Mg2+-dependent step (the E~P to EaP transition) which separates the two halves of the stepwise mechanism (reaction scheme !). Nevertheless, we suggest that great caution is needed in the interpretation of these results. The key point in the mechanism is that the phosphorylated intermediates are considered to be stable unless dephosphorylation is brought about by A D P (acting

153 on EIP) or by K ÷ (acting on E.,P). In order to see the stable intermediates, Na +dependent phosphorylation should be measured in the absence of A D P and K ÷, but as it is difficult to remove adventitious K ÷ some spontaneous breakdown is to be expected and is, in fact, observed. Obviously, provided it is slow, the spontaneous turnover can be ignored [24]. However, the logarithmic scales used for plotting the concentrations of the phosphorylated intermediates tend to conceal a rather rapid spontaneous breakdown, possibly exponential, even when only adventitious K ÷ is present, and this is harder to ignore (on this point, see also Section ILIA-3). The stability of intermediates is so crucial to the stepwise mechanism, that if stable intermediates have not been proved to exist, then this throws doubt upon the mechanism itself. This means that we need to know more about how K ÷ interacts with the system. Fresh light on the role of K ÷ comes from an important and rather neglected study of the phosphorylated intermediates by Tonomura et al. [26-28]. In the first two papers, their results with Na ÷ and K +, with particular reference to the hydrolysis of phosphoprotein, did not show the apparently clear-cut effects previously described. They put forward the following reaction scheme [26,27].

E*ATP~

Mg 2° Na ÷ ~ EI"ATP~-~E2.ATP Mg 2°

,,

~

K*

K*

EP

~ E* P i

(3)

NO*

Their salient new finding was that the phosphoprotein is formed after, and not before, the reaction with K +. More recently, K ÷ was shown to have dual effects on the phosphoprotein [28] in inducing breakdown not only by hydrolysis to give Pi but also by regeneration of ATP. Another finding, at variance with the stepwise mechanism was that the sensitivity of the phosphoprotein to A D P occurred at a later time than sensitivity to K +. A rather similar result, irreconcilable with the straightforward stepwise mechanism (reaction scheme 1), is that K ÷ can stimulate A T P - A D P exchange [29]. The main conclusion is that there are dual effects of K + such that the intermediate can be hydrolysed or take part in transphosphorylation to A D P [26-28]. Another set of results cast doubt on the sequence of effects of Na ÷ and K ÷ on the reactions of the phosphoprotein [30-32]. Before ATP can be hydrolysed it must become bound, and the features of the binding in relation to the effects of Na ÷, K ~ and ouabain give information about the steps at which these ions act. It was found that (i) there is no ouabain-sensitive ATP binding unless Na ÷ and K ÷ (as well as Mg +2) were present and (ii) the pH profiles for ATP binding and ATP hydrolysis are superimposable but different from the pH dependence for phosphorylation. These important results show a requirement for K ~ at the very first stage in the reaction, and an inconsistency in the pH effects such that it is necessary to exclude the phosphoryfated intermediate as being involved in ATP hydrolysis when both Na + and K + are present [30-32]. IB-2. The Bhtding of Pl. The second feature of the cyclic reaction scheme 2 is

154 that Pj can become covalently bound to the enzyme by reversing the last step of the reaction. There is no doubt that with intact cells, there can be reversal of the overall reaction, but this is a quite different system from fragmented membranes, in which it is claimed that there is reversal of a partial reaction unconnected with ion gradients. When the normal cation gradients are reversed, the pump can be made to run backwards so as to synthesise ATP from ADP and P~ with accompanying ouabain-sensitive ion movements [33-35]. The free energy needed to form ATP is derived from the cation gradients, just as in vivo, the free energy of hydrolysis of ATP is used to generate the cation gradients. To form phosphoprotein, a source of energy must be available, yet what could this be with fragmented membranes? There are no ionic gradients and no high-energy, low-molecular weight energy donors. It therefore seems unlikely that a low-energy compound like P~ could react with protein to become covalently bound and give the same product as that obtained with ATP. Nevertheless, in spite of theoretical thermodynamic misgivings, there does seem to be good evidence for the reaction [36-38]. It should be emphasised that, in contrast to work with intact cells, the incorporation of P~ into protein, as opposed to ATP, has been obtained in particulate systems where there is apparently no vectorial component. The question at issue is not whether the overall reaction can be reversed, but whether P~ can be incorporated into an acyl phosphate bond in a protein with preparations of fragmented membranes. The possible alternative explanation, that the observation depends on part of the preparations actually existing in the form of vesicles, should be considered. When vesicles are present, conditions can easily arise for a variety of modes of operation of the pump to occur in the same preparation (see Section IVB-I-4). Vesicles can be inside out or the right way round as regards orientation in the membranes and the results will then be different from those with preparations of truly fragmented membranes. Under appropriate conditions P~ could be incorporated into protein as described, but presumably only when energy from an ionic gradient is available. Careful tests in this laboratory with a preparation lacking vesicles have shown satisfactory phosphorylation of protein with Na ÷ ; ATP H- Mg 2÷, but no indication of covalent incorporation of inorganic phosphate under a variety of experimental conditions (D. Wattam and R. Whittam, unpublished observations). In particular, addition of ouabain and M g 2+ did not stimulate P~ incorporation even though ouabain itself was bound at a satisfactory level. Since ionic gradients clearly do not exist with truly fragmented membranes, it is perhaps not surprising that we find no P~ incorporation, yet at the same time there was good (Na + -- K+)-ATPase activity. We believe that incorporation of P~ into protein does not occur in a scalar (i.e. non-directional) reaction of the ATPase. There is as yet no evidence on the ionic gradients that may be required in vectorial systems for Pi incorporation and as regards the reaction mechanism of the ATPase, it seems premature to draw conclusions. IB-3. Quabain Binding. The third aspect in the cyclic scheme is that ouabain binds only to E2 conformations. There is certainly much evidence pointing to two

155 kinds of pump conformation, one of which binds ouabain and one which does not [23-25]. Moreover, it is clear that there are two ouabain binding pathways, one requiring Na ÷ + ATP -[- Mg 2+ and the other requiring Mg 2+ but inhibited by Na ÷ [39]. In both pathways K ÷ inhibits the binding. However, the cyclic scheme shows no reaction pathways whereby Na + or K + would convert E2 conformations to E~ conformations and thereby inhibit the binding. A further difficulty is that ouabain can become bound under conditions which do not allow formation of the ouabain-enzyme complexes shown in the cyclic scheme [23,39] The effect of Pl on ouabain binding has had much significance regarding the pump mechanism. It has been said that P~ stimulated ouabain binding, consistent with its incorporation into protein [23-25, 36-38]. There is a conflict of experimental results in this connexion, however, and here again the discrepancy may arise because of the different properties of the preparations used. In our hands, there is very little influence of P~ on the level of ouabain binding [39,40]. Placing particular emphasis on the quantitative aspects, we measured ouabain binding to an enzyme preparation from ox brain in the presence of Na + ,'-- ATP ,'-- Mg 2+, Mg 2+ alone and Mg 2÷ + Pt. Sufficient time (1 h at 37 °C) was given for the system to reach equilibrium. The results showed that, in contrast to earlier non-equilibrium studies, the binding with Na + -- ATP -!. Mg 2+ was quantitatively equal to the binding with Mg 2÷ alone. Pi raised the binding in the presence of Mg 2÷ alone but only by about 2 5 ~ [39,40]. Moreover, the similarity in the binding of ouabain with Na + ÷ ATP ÷ Mg 2÷ and Mg 2+ alone and the small effect of Pi has been confirmed with enzyme preparations from rabbit kidney and guinea-pig kidney and brain in this laboratory (C. Hallam and R. Whittam, unpublished observations). Thus, the evidence is rather uncertain and probably depends on the kind of preparation and how truly fragmented the membranes are. Until clearer results are presented, ideally with a soluble preparation, for P~ incorporation and for stimulation of ouabain binding by Pl, it seems premature to base the mechanism on the reaction shown at the bottom of the cyclic scheme. Another feature casting doubt on the cyclic reaction scheme is the pH dependence for ouabain binding [39]. The binding in the presence of Na + -~- ATP ÷ Mg 2÷ was insensitive to pH over the range studied. In contrast, the pH profile for ouabain binding in the presence of Mg -'+ alone was superimposable on the pH profile for overall ATP hydrolysis. The phosphorylated intermediate cannot be formed with M g 2÷ alone but requires Na ÷ 7 ATP -~- Mg, 2÷ yet it is under the latter conditions that there is the discrepancy in pH dependence. This comparison of the effects of pH on ouabain binding and on ATP hydrolysis therefore suggests that the phosphorylated intermediate is not required for ouabain binding and is unlikely to be part of the normal reaction sequence [39]. IB-4. The hlfluence ofpH. Perhaps the main finding that raises doubts as to the validity of the stepwise mechanism is that K ÷, as well as Na ÷, is required at an early step in the reaction. We have previously developed the view, from other evidence, that Na ÷ and K + are required together [39]. The features of the partial reactions should be consistent with the properties shown by the complete system, for

156 example, as regards pH dependence. However, this requirement has not been met. The pH dependence for ATP binding, ATP hydrolysis and the phosphorylation reaction has been measured [30-32]. ATP binding was sensitive to pH in the same way as overall ATP hydrolysis. In contrast, the pH profile for the phosphorylation reaction was different, and there was considerable phosphorylation at pH values, where overall ATP hydrolysis was low. The partial reactions would seem not to bc parts of the overall reaction, but rather side reactions, which cannot be put together to give the main pathway for ATP hydrolysis and for active transport. IC. The Transition State Mechan&m It is now worth considering the evidence for an alternative mechanism o f a "one-step" reaction for ATP hydrolysis, in which there is simultaneous binding of internal Na +, external K + and A T P ' M g 2~ [39]. The enzyme-substrate complex that is formed is converted to a transition state complex, as the reaction takes place. The reaction sequence can be represented as follows: INSIDE

No* Pump K* OUTSIDE

Pi

AT P Mg 2.

~-- P u m p - A T F L M g

I

;

~.- P u m p

Iump.

k

(4)

_1

The sites must have high specificity and high affinity for the ions to become bound in the enzyme-substrate complex. However, the chemical specificity is not absolute because there can be competition between Na t and K +. in the transition state complex, there is transient incorporation of phosphate from ATP causing displacement of the ion binding sites, perhaps over only a few Angstrom units, such that the ions are moved in position. The complex contains K + and therefore decomposes spontaneously. This breakdown leads to a fall in the affinity of the ion binding sites so that Na + and K + are released. The complex can only be formed and hence decay when the sites are properly and correctly occupied by Na ~ and K + [39]. It must be emphasised that a transition state is an entirely different concept from an intermediate. An intermediate is a stable compound, which can be isolated under suitable conditions. In contrast, a transition state complex is the unstable species, which can never be isolated and which is interposed between the reactants and products in every chemical reaction. It has a distorted or strained structure, which is not quite like either the reactants or the products and so it cannot be formulated in the same way as a chemical compound. Obviously, the transition state shown here conforms to this criterion. The conformational change leading to an intramolecular shift in position of Na + and K + is generated by the transient incorporation of phosphate from ATP. The formation of the transition statc complex requires the combined action of Na ÷, K ~ and ATP • Mg 2+ and its breakdown is an inevitable consequence of its formation. Thus, the transition state, as the intermediate in the reaction, is a

157 fundamentally different entity from a phosphorylated intermediate. Nevertheless, there are superficial similarities between the transition state complex and the E_~P phosphorylated intermediate, since in both materials the Na + binding site faces outwards and K ÷ causes hydrolysis. The question therefore arises whether a new line of experimental evidence can be adduced, which will allow a clear distinction between the stepwise mechanism and the transition state mechanism. The crucial difference relates to the action of K ~, especially whether its activation is concomitant with or later than Na +. This difference is so pivotal that we shall dwell upon it throughout the remainder of this article. In order to approach the problem in a new way, we have turned to conventional kinetic analysis as a means of resolving this question.

11. KINETIC ANALYSIS OF THE REACTION SEQUENCE Elucidating reaction sequences is a problem common to all mechanistic investigations of multi-substrate enzymes and we regard Na + and K + as substrates [41-50]. In broad terms, enzyme mechanisms fall into one of two classes which formally correspond to the two types of system envisaged for the pump, and exemplified by the stepwise and transition state mechanisms. Thus, the stepwise mechanism is classified as "one-unit" because it binds only one ion at a time and has the features of a "'substituted" or "ping-pong" enzyme system. The characteristic feature is that one or more product-release steps precede one or more of the substrate-binding steps. Thus in the stepwise mechanism, A D P plus external Na + are considered to be released as products before K + binds and P~ is released. O,a the other hand, the transition state mechanism for the pump is classified as "two-unit'" because the two ions (Na + and K-) are bound simultaneously corresponding to "sequential" enzyme systems. The two kinds of mechanism can be, and in very many cases have been, distinguished by studying enzyme kinetics and more detailed reaction sequences within each class can be worked out by the same technique [41-50]. Before turning to kinetic studies on the pump, it must be pointed out that in the nature of the experiments, evidence proving a mechanism cannot be obtained. The results can only be consistent with a mechanism. Moreover, each result may have more than one interpretation and it is often necessary to supplement "'initial velocity" studies with other kinetic tests [41-43,50]. Nevertheless, a positive aspect of kinetics is that mechanisms can properly be rejected however plausible they may be on other grounds.

11,4. Tile Enzyme-Substrate-Modifier Analysis All the conventional kinetic studies on the pump have made use of the general enzyme-substrate-modifier treatment which has been described in detail by Segal, Katchmar and Boyer [44] and by Frieden [48]. The procedure has been to examine the sequence amongst pairs of substrates, which may be Na - and ATP, K + and ATP

158 or Na + and K +, with the third held constant, by measuring the activation of the p u m p by one substrate (S) at different fixed levels of the other (M) [51-55]. Pump activity has been measured variously by ATP breakdown or ion movements. The reaction scheme in the general case is [48] Kl

E

-~

K2

E

-- M = = E M

Ks

ES + M = EMS

K4

EM -~- S == EMS

S

=

ES ES

-k~products

(5) EMsk-~oproducts

The modifier may inhibit or activate, but for the present purpose it is taken to be a second substrate and so the rate constant k5 approaches zero. The rate equation for the general reaction, where the modifier (M) and the substrate (S) bind in a r a n d o m sequence with affinities defined by the dissociation constants Kj to K4, is therefore v Zr---

1

+

k6/(1 +- K3/M ) K, / 1 + M/K2" I

S

(6)

~ 1 --, M / K a I

It follows from this equation that plots of l/v against I/S at different fixed levels of M may intersect on the left of the ordinate or be parallel according to the relative magnitudes of the four dissociation constants, K~ to/(4. In order to test for sequences one of the constants is assigned a value of infinity : this has the effect of blocking the reaction pathway in question. For example, consider the case of an ordered reaction when M binds before S, K t iS infinity and K 3 must become zero. Equation (6) can then be arranged into the form I' --

](6 =

"

E

K4

(7)

I - : " - - i f - (1 -~ Kz/M)

By comparing it with the simple Michaelis-Menten equation, v

V :.........

,'¢.,

1

(8)

S

it is easy to see that in this case V is independent of M and therefore so too is the intercept on the ordinate in the Lineweaver-Burk plot (l/V). It follows that if M binds before S, plots of 1/v against 1IS at different levels of M will be a family of lines intersecting on the ordinate. This is one of the tests we have applied where M is Na + and S is K + [55]. (Since the results did not give lines intersecting on the ordinate, we conclude that N a - does not become bound before K ÷, see section lIB-3).

liB. Kinetic Tests HB-I. Sequences hlvolving ATP. From the practical point of view, the easiest test of the sequences are for Na + and A T P and for K + and A T P in a fragmented

159 membrane system. The disadvantage is that the vectorial feature of the system is lost. This means that ion movements and hence also the sequence for Na + and K ÷, cannot be examined and that mutual competition between Na ÷ and K ÷ is unavoidable. Two attempts have nevertheless been made with particulate preparations to elucidate the order in which Na ÷ and ATP" Mg 2÷ react [51,53]. The effect of Na ÷ (as modifier) was examined on the activation of hydrolysis by ATP (as substrate) with K ÷ held constant [51]. The results were analysed as described by Frieden [48] and suggested that Na- and ATP bind in a random order, and the same conclusion was reached in a somewhat similar and more recent study [53]. The sequence for K ÷ and ATP was examined in a comparable manner. However, there was a discrepancy in the interpretation of the results by these two groups as to whether K + binds before ATP (Eqn 7) [53] or randomly (Eqn 6) [51 ]. In any event, a reaction of K + after Na ÷ release is excluded by these findings. In this connection, these results, whatever their interpretation, are in line with the view that K ÷ is required for ATP binding [30-32]. HB-2. The Independence of the Na + and K + Binding Sites? The question now arises whether the more interesting and important sequence for Na + and K + can be determined. Here an intact cell system is essential because of the necessity of avoiding competition between Na + and K + at the same side of the membrane and the necessity to separate products and substrates. In contrast to the work described in the last paragraph, one of the ions is treated as substrate and the other as modifier with A T P ' Mg z+ held constant. Pump activity can then be measured either by the rate of movement of the substrate (K +) or by the rate of ATP hydrolysis. It is clear that these measurements are only possible so long as the product can be distinguished from the substrate; when these are the same chemical ion, the distinction depends on the spatial separation across the membrane. The substrate becomes the product as it is transported from one side to the other. An unequivocal distinction can be drawn between one-step and two-step reactions in experiments, in which spatial asymmetry is maintained provided care is taken to eliminate competition. In the one-step mechanism it will be apparent that a change in concentration of modifier should affect the affinity of the enzyme for the substrate, shown by a variation in the Km for S as M is changed (Eqn 6): the associated changes in V have also to be taken into account. Two pieces of work bearing on this point have been described [52,54], but in both the kinetics of the system was not studied from the point of view of elucidating the sequence for Na ÷ and K ~-. Thus, whilst the results of both allow for the possibility of a one-step reaction, the experiments were not designed so as to rigorously exclude a stepwise mechanism. It is important to show why these results are equivocal and why our own work became necessary, to answer the question. Experiments on K ÷ influx and Na ÷ efflux with sheep and human erythrocytes have been interpreted as showing that the affinity for each ion at its own class of sites was independent of the degree of saturation of sites belonging to the other ion. on the opposite side of the membrane [52,54]. Both these studies lead to the view that the reaction of an ion at the site is unaffected by occupancy of the other site, i.e. the sites

160 ~3re independent of each other. The agreement in these two studies, whilst impressive, must be regarded with caution because of a fundamental feature in the experimental set-up with regard to competitive inhibition. There must have been competition between Na t and K ÷ either within the cells in one study [52] or outside in the other [54]. The complications arise from the fact that ions of the same element are both activating and inhibiting according to location. For example, activation by external K ~ is ~.ith cclls in which internal K ~ is interfering with the effect of internal N a ' , and yet it is the influence of internal Na* that is the crucial point at issue. Competition is bound to distort the results from the ideal pattern associated with the kinetic equations for the activating ions. Thus the results could represent a special case of ~he general rate equation (6)where Kj :: K4 and K2 ~ K3, but in view of the existence of competition, they could represent an ordered reaction or even a two-step reaction [50]. lIB-3. Evidence for the Random Attachment o f Na t and K ' . In order to avoid these impediments to interpretation, competition was excluded in two studies carried out in this laboratory. In the first set of experiments, A T P hydrolysis in resealed erythrocyte ghosts was measured with external K" as substrate at two different levels of the modifier, internal N a ' . Choline was employed as the major replacement cation inside and outside [55]. A characteristic kinetic pattern was revealed and it was therefore necessary to do the corresponding experiments with a more direct method lk~r estimating p u m p activity, namely net K + influx (A. R. Chipperfield and R. Whittam, unpublished observations). The results of both studies showed that the two lines in the Lineweaver-Burk plot were not parallel but intersect to the right of the ordinate. In a typical experiment on increasing internal Na" from 4.5 mM to 27 raM, there was a rise in the K,,, for external K ~ from 20 tCM to 65 ~uM. In intact cells leakage of Na" and K + from the cells to the medium is unaw)idable, but we showed that the leakage was too small to account for the apparent changes in affinity [55]. Therefore there is an interaction between the two ion-binding sites. Eliminating competition between the ions at both surfaces of the membrane seems to be the indispensible experimental condition, which allowed the change in affinity to be seen. These results exclude an5' two-step p u m p mechanism and any ordered one-step mechanism. If k5 is not zero but tinite, but otherwise negligible, it follows that the results are consistent with r a n d o m addition of Na * and K ". This will only be true if, in the general enzymesubstrate-modifier scheme [48] (Eqn 5), K~ -~ K,~. This scheme, translated into a form appropriate for the pump, is shown in Fig. I and from this scheme it is possible to begin to see how the observed changes in K,, and V, otherwise revealed as a pattern of lines in the Lineweaver-Burk plot, can arise. When internal Na" is raised, the concentration of E N~ will be raised and this will enhance the flow through the right hand pathway. In this pathway, as Kt -:-~ K,,, the affinity for K + is low and therefore raising internal Na ~ will have the effect of raising the Km for external K '. In addition, raising internal Na + will raise V and this will appear as an intercept effect in the Lineweaver-Burk plot. Thus, it seems reasonable to conclude that the results show that Na ~ and K ~ bind to the pump in a random sequence [55].

161

OUT K

Na

Fig. I. The general mechanism of Frieden [48] for a substrate and modifier (external K- and internal Na ÷ respectively) is shown in the form applicable to the sodium pump. The constants K~ and /(4 are dissociation constants. The constant k e is the rate constant for the breakdown of the enzymesubstrate-modifier complex is measured experimentally as the rate of ouabain-sensitive Pj production [551 or net K ÷ influx. (The velocity in the absence of modifier (external Na ÷) is zero: thus k 5, which would be the rate constant for the breakdown of the enzyme-substrate complex (Eqn5) is negligible and does not appear here or in Eqn (6)). ATP must react before the transition state can form but the points of addition of ATP and liberation of ADP are not specified here. The transition state complex K+ is shown as [EP~,÷] and the square brackets imply, as in chemical mechanisms, its unstable character [39]. For simplicity, in the scheme shown, the numbers of Na" and K ~ transported per phosphate ion produced are not indicated (see Section IVB-4). Figure reprinted from ref. 55 by permission of the Royal Society.

HC. Limitations and Scope of the Kinetic Approach W e m u s t n o w r e c o g n i s e that, in a d d i t i o n to the p r a c t i c a l difficulties a l r e a d y m e n t i o n e d , t h e r e is the m a j o r t h e o r e t i c a l c o n s i d e r a t i o n

o f w h e t h e r the e n z y m e -

s u b s t r a t e - m o d i f i e r analysis c a n p r o p e r l y be a p p l i e d to the p u m p .

T h i s analysis [48]

lies b e h i n d all t h e kinetic studies d e s c r i b e d here [51-55]. T h e t h e o r e t i c a l difficulty arises b e c a u s e m o r e t h a n o n e ion o f e a c h kind c a n i n t e r a c t with the p u m p .

To

illustrate the p r o b l e m , s u p p o s e t h a t t w o N a + a n d o n e K + react with the e n z y m e ; if the g e n e r a l rate E q n (6) is e m p l o y e d to e x a m i n e the s e q u e n c e for N a + a n d K +, it will t a k e i n t o a c c o u n t the sequences, Na +Na + K +orK ~,

+

~,

+Na +Na ÷ ~,

~,

~,

or, for r a n d o m b i n d i n g , b o t h .

(9) But it will n o t t a k e into a c c o u n t the m i x e d s e q u e n c e

Na ÷ K + Na +

T h i s difficulty b e c o m e s e v e n m o r e a c u t e if t h r e e N a + a n d t w o K + react w h e n n o less t h a n 5! = 120 s e q u e n c e s are possible.

S u c h c o m p l e x i t y is far b e y o n d the s c o p e o f

162 any available kinetic equation. For example, even the empirical rate equation for a three substrate (A, B. C) reaction in which e is the concentration of active centres, Vothe initial rate and ~oo, ~A, etc. are functions of dissociation constants e vo -- ~°° +

~PA _t_ ~:t~ . [A] [B]

~°c _t. ~VAB , CFAC CFt~C [C] [A][B] [A][C] -1 [B}[C]

~FABC [A}[B}[C]

(11)

applies only if the reaction shows Michaelis-Menten kinetics with all three substrates [49]. For the pump, this is certainly not so, in view of uncertainty about the nature of the activation mechanism for the ions. If more than one Na* or K ÷ bind, plots of l/[Na] or l/[Na] 2 etc. should be curved [50] and when such deviation from MichaelisMenten kinetics is seen, more complex analyses are required [54,56]. Of these the simplest [54] is the rate equation V v

--

-

-

(12)

This can be transformed into a linear form (by plotting S/v I ;n against S), akin to a Hanes plot [41,57]. However, particularly with activation by external K ÷ with no external Na ÷ present, no deviations from Michaelis-Menten kinetics can be seen [55,58]. When this happens, it is then proper to accept that the activation is hyperbolic and to base the kinetic analysis upon this assumption. This is true even though one may suspect that, under these conditions, the deviations are there but too small to see. To a very large extent, the technical and theoretical problems may be overcome by applying Cleland's "slope and intercept" analysis [47]. For example, with particulate systems it is reasonable to assume that A D P and Pi are at zero concentration. It follows that there would be no reversible connection between the two halves of the stepwise mechanism (reaction scheme 1) and therefore that competition between Na ÷ and K ÷ would affect intercepts but not slopes [47]. lndeed the observed deviations from the predictions of this mechanism do show that the stepwise mechanism can be excluded. Moreover, by the same analysis it can be shown that this exclusion remains true even if more than one ion of each element add to the enzyme [47]. Similarly, mixed sequences like Eqn (10) can be taken into account and excluded. Thus, although some fault may be found with the kinetic studies described here, the exclusion of all "'two-step" mechanisms and nearly all ordered "'one-step" mechanisms in every one of these studies cannot be lightly set aside [55]. Obviously, these results [51-55] are consistent with the transition state mechanism, but so far we have said nothing about how ATP-Mg 2+ is considered to interact with the system or how ion movements, other than coupled pumping of Na* and K*, may be accomplished. In the next two sections we shall discuss these two points. i11. THE KINETIC BASIS FOR AFFINITY CHANGES The kinetic approach to mechanism is to measure how one substrate influences

163 the kinetic constants (K,, and V) of another substrate [41-50]. We shall now consider further implications of this approach, particularly since the changes in affinity which appear to occur with the pump have already been the subject of much discussion, the object being to disclose the reaction mechanism [9-16]. HIA. Mutual Effects o f K + and A TP H I A - I . The A T P a s e reaction. A direct connexion between the sites for K + and

ATP is suggested by the fact that the Km for ATP of about 0.3 mM under optimum conditions for hydrolysis was quite different from the K,, of 1/~M in the phosphorylation reaction where no K + is added [19]. The question arises whether the reaction mechanism can give rise to this affinity change. This possibility has been recognised [59] and discussed more specifically in a detailed account [60]. The relevant observation was that a plot of I/v against 1/ATP was a family of parallel lines at different levels of K + [59] and it was shown, by deriving the rate reaction from Briggs-Haldane kinetics, that this kinetic pattern could arise from a two-step reaction [60]. This analysis can account for the changes in ATP affinity caused by K + but there were two reasons for the belief that the analysis was incomplete and therefore of doubtful significance [60]. First, the parallel pattern could arise in several ways, one of which is random addition of K + and ATP [48,51]. Second, there was the complication arising from the Na÷-dependent, ouabain-sensitive ATPase which is seen at low ATP concentrations and which instead of being activated by added K ÷, was actually inhibited [61]. ILIA-2. K + as a Product Inhibitor. This last observation has been inexplicable. However, a clue is given by the fact that just as lowering K + raises the affinity for ATP, a converse relationship can be predicted. This is that lowering ATP raises the affinity for K +, as has been shown experimentally [62]. The shift in affinity was so large that the adventitious K + was sufficient to activate the Na+-dependent, ouabainsensitive ATPase suggesting that the reaction was none other than the normal pump ATPase, which depends on mutual interaction of Na +, K + and ATP. It is significant that the amount of extra K + needed for inhibition is far less than that necessary for K + to compete with Na +. Such an effect is comparable to the product inhibition, which has been described for other enzymes [46,50]. The action of K + as a product inhibitor will arise when the equilibrium, E • Product = E + product

(13)

is not displaced entirely to the right. Normally, this would not be expected to occur except at very high K + concentrations, but the shifts in K + affinity allow it to happen when the ATP concentration is low. According to this line of reasoning, it follows that as the ATP concentration is raised, there will be a progression towards higher K + levels for bringing about product inhibition. This effect has been demonstrated experimentally. With 0.9/~M ATP, added K + began to inhibit at about 0.03 mM but with 13/~M ATP, added K + first stimulated the reaction up to 1 mM and inhibited only at higher concentrations [63]. These results show that K + directly inhibits ATP

164 hydrolysis as well as activating. Thus, there are three actions of K ~: K* can activate synergistically with Na +, it can inhibit by competing with Na ~ and it can inhibit directly as a "'product inhibitor". ILIA-3. The Spontaneous Turnover of the Pho.whorHated Intermediate. In connection with the fact that the effects of traces of K ' can be important, it is interesting that the N a ' - d e p e n d e n t ATPase not requiring added K + is observed under the same conditions as Na ~-dependent phosphorylation with ATP. Since this ATPase activity depends on bound K +, the possibility arises that this bound K" can cause spontaneous turnover of the phosphorylated intermediate. This seems very likely, for the turnover of the intermediate resembles the turnover of a typical transient even though the turnover is some 100000 times slower than with optimum K ~ [43,64].

IHB. ITP and UTP Hydrolysis The hydrolysis of ITP and U T P is also stimulated by Na + and inhibited by K + [65,66]. It is most economical to conclude, as with the Na ,-dependent ATPase, that these reactions are activated by adventitious K + and that added K + acts as a product inhibitor. If so, then why is the affinity for Na t lower with ITP (or UTP) as substrate than with A T P and the affinity for K t so much higher'? The key point is that ITP and UTP themselves bind much more weakly to the enzyme than ATP, and their K,, values are higher than for A T P [65 -67]. The apparent Km for another substrate with A T P as moditier will be (from Eqn 6) K, •

(1 -i- M/K2) (1 -t- M/K3)

(14)

Clearly, the K m depends upon the relative magnitudes of the four dissociation constants (Eqn 5) and without knowing these in every case, it is not possible to predict how replacing A T P with ITP or U T P would shift the K.,, for the substrate. Experimentally, the results show that the effect is to lower the affinity for Na + and raise the affinity for K ~ [65,66].

IIIC. The Role of Mg 2~ In addition to Na ~ and K +, the divalent ion M g 2- iS also an essential requirement for the A'l'Pase [5]. The mode o f action of Mg 2~ has a bearing on the mechanism [9-16] because according to the stepwise mechanism, Mg 2" binds so as to promote the E I P to E2P transition, and it is thought that changes in affinity for Na + and K - reflect this feature. The key question is whether the sodium p u m p ATPase consumes the substrate as free A T P or as the A T P " Mg z+ complex and there are conflicting views on this point. However, the results obtained from other ATPases show that almost without exception they hydrolyse the substrate in vitro in the form of the A T P • Mg 2+ complex and this is the form of A T P available within the cell [50]. Moreover, the evidence on this point is quite clear: the proper test is to calculate A T P - Mg 2+ concentrations and to see whether activation by this species follows

165 Michaelis-Menten kinetics [50]. This test has been applied in three laboratories with particulate enzymes from rabbit kidney [68], rat brain [51], and human erythrocytes [53] and the results agree in showing that the true substrate is the A T P " Mg 2+ complex. As with other enzymes [50], free Mg z+ and free ATP inhibit [69] (S. A. Wade and R. Whittam, unpublished observations). Also consistent with ATP • Mg z+ being the substrate, as opposed to free ATP, is the finding that Mg 2+ is needed for ATP binding to the pump [30-32]. Apparently against this view are the observations that free ATP and free Mn 2~- bind to the enzyme [67,70,71]. However, this is consistent with the view that binding of the purine moiety and the Mg 2+ • phosphate moiety of the substrate requires attachment at separate regions in a single site [43,50, 67,71]. Moreover. these results on ATP binding are in line with the work on ouabain binding supported by ATP [23,39]. Thus it seems that a system with one site for the ATP • Mg complex and with random attachment of Na + and K + must inevitably account for many of the apparent complexities of the sodium pump.

IV. THE BRANCHED TRANSITION STATE MECHANISM So far, we have considered only how the transition state mechanism can account for coupled ion movements associated with the normal physiological operation of the sodium pump. We have shown that the kinetic analysis is consistent with this mechanism [51-55]. However, there are various modes of operation both as regards ion movements and the chemical reactions, and any mechanism, if it is to be satisfactory, must account for these. For example, there is Na+: Na + exchange in the absence of external K ~. One way to explain this phenomenon would be if external Na + could be pumped in the same way as external K ~. If this were so, ATP should be hydrolysed but it is not [72]. Evidently the specificity of the enzyme is such that unless the ion-binding sites are properly occupied by Na + and K ~ no chemical reaction will occur and the transition state will not be formed and cannot therefore break down. In the absence of external K +, the stable phosphorylated intermediate will be formed and catalyse Na+: Na + exchange without the production of Pi. The questions we now consider are how the transition state mechanism may accommodate some of the various modes of operation of the pump.

IVA. The Definition of the Branched Mechanism A unique feature of the transition state mechanism is that the ion binding sites for Na + and K + areshifted in position so as to face the opposite sideof the membrane before dephosphorylation may occur [39]. The question arises therefore whether the sites could be accessible frown that side. Thus, if the K+-binding site was not already fully occupied by a saturating quantity of external K +, could it react with internal K + to form the same intermediate as usual? The transition state would be the same regardless of how it had been formed, whether by loading the K-binding sites from

166 inside or outside, and thus form the same products. On the other hand, if one or other substrate (say K ÷) were missing the transition state would not be the same and accordingly it must either revert to the original state or form a different product (the well-known phosphorylated intermediate). As a consequence of the characteristic feature of the transition state mechanism, it can be modified into what will be termed the branched transition state mechanism. The branched mechanism is formulated so as to account for the modified ion movements and modified chemical reactions which can be observed when conditions are not optimal for physiological operation of the pump owing to the absence of either N a + or K +. It is more satisfactory to show two separate diagrams. The ion movements may be represented as follows, The u n c o u p l e d pathwGy activation by i n t e r n a l K

~ E~No~ . . ~

ENo~

products

E O U ~ E K S K

(15)

' y

•

Activation by external K the coupled pathway

This formulation, omitting the number of Na + and K + reacting, is not to be taken as necessarily implying that the pump can function normally when there is no activation by external K +. The partial reactions are; No

Mg

f

~_ E NO

IP

~- E-Mg

IN

~.

E P

ou o ba ~n - e n z y m e - corn plexes

Na, K

EOUT

k~

Phosphorylation

Na EK

NOoK

r-~q ATP,Mg ~_/E~pI

.~

products

LNo] (16)

K

~_

EK

pNPP

~- pNP • Pi

167 We shall now discuss how the branched transition state mechanism fits. IVB. The Evidence f o r the Branched Mechanism IVB-I. Internal K +, its Role in Ouabain Binding. Ouabain binding to erythrocyte ghosts has been shown to be inhibited by external K +. A new and unexpected finding was that in the absence of external K +, there was inhibition by internal K + and this could not be attributed to leakage [73]. How can internal K + influence ouabain binding to the external surface? An explanation readily emerges from the branched transition state mechanism (reaction scheme 15) in which the K + binding sites faces inwards and can then become loaded with K +. IVB-2. blternal K +, its Role in Na + : Na + Exchange. Na + : Na + exchange is ouabain-sensitive and mediated by the pump provided there is no external K + [72]. As external K + is raised, N a + : K + coupled ion movements begin in place of Na + : Na + exchange. This reciprocity is consistent with a one-step system, but it is difficult to explain in a two-step system [14,15]. A further interesting point is that the Na + : Na + exchange that is seen in the presence of ATP shows a requirement for internal K ÷ [15,72]. It follows that this exchange takes place via the uncoupled pathway in the branched transition state mechanism (reaction scheme 15). IVB-3. Internal K +, its Role in K + : K + Exchange. The pump can also catalyse K + : K + exchange. An experimental analogy with Na + : Na + exchange is that it is strongly inhibited by internal Na ÷ and again it would appear to be inhibited to the same extent as coupled Na + : K + transport is activated [74]. On the other hand, K + : K ÷ exchange and Na + : Na + exchange differ inone important respect. The affinity for external Na + in Na + : Na + exchange is extremely low [54], whereas the affinity for internal K + in K + : K + exchange is apparently high [74]. This suggests that K + : K + exchange does not represent total reversal: rather, it is accomplished by a reaction of internal K + with ENa, followed by reversal of the normal translocation step (reaction scheme 15). IVB-4. FleMbility in Na+ : K + Coupling. Work on erythrocytes appears to show that the coupling of the pump is constant, at 3 : 2 : 1 for Na + : K + : ATP respectively [75-77], but work with excitable cells shows that the Na + : K + ratio may vary. When internal Na + is raised, the system pumps proportionately more Na + and less K + [78,79], giving the electrogenic effect. In the branched mechanism (reaction scheme 15), if internal Na + is raised, the relative amount of E Na will increase and since it is from this intermediate that the uncoupled pathway branches, this will have the effect of enhancing the reaction rate in that pathway. Thus, as internal Na + is raised, the pump becomes uncoupled and therefore electrogenic. IVB-5. Ouabain and Oligomyein Sensitivities. Another striking feature of the branched mechanism is that it provides a rational basis for the different sensitivities of various reactions to ouabain and oligomycin [9-16]. For example, phosphorylation, A T P - A D P exchange and p-nitrophenylphosphate hydrolysing activity are much less sensitive to ouabain than either ATP hydrolysis or ion movements. Oligomycin does not inhibit the branch reactions, whereas both A T P hydrolysis and ion

168 transport are inhibited. scheme 15)

Thus, oligomycin inhibits only the reaction (in reaction

EN,, K - - } " E- K ~

(17)

Further support for this view comes from studies on p-nitrophenylphosphate hydrolysing activity, which can be activated either by K + alone or synergistically by Na + plus K +. When it is activated by K ~ alone oligomycin does not inhibit but when it is activated by Na + plus K ' it does [80]. On the other hand, the significance of the phosphatase is not at all clear, for there is considerable doubt concerning the vectorial aspects of this reaction and wb.ate~er ion movements may accompany it [81--83].

IVC. Tile Phy,siological Role of htternal K' We have already explained how internal K ~ can regulate p u m p activity through its action as a competitive inhibitor with internal Na- and as a direct product inhibitor (see Section IliA-2). The question may now be asked whether these are the sole physiological effects in view of the claim that Na ~ efflux is stimulated by internal K + [54]. Na ~ effiux was measured at different internal Na* concentrations with internal K~ varied over a wide range. In some experiments, the internal K + was very low and it was then that Na ~ efflux fell as internal K ~ was decreased [54,84,85]. K ~ inllux also fell in a like manner. The suggestion was that internal K ~ can stimulate the pump. It is important to note that in this work there was enough external K + to saturate from the outside. An alternative and more likely explanation should be considered. When internal K ~ is lowered the supply of A T P from metabolism is reduced and this in itself would cause a fall in p u m p activity [86], and with less than 20 mM K ~, it cannot be assumed that the A T P supply from glucose metabolism remains unaffected. We believe that in the presence of saturating external K +, the sole influence of internal K ~ is to inhibit and that this is the means by which the p u m p is physiologically regulated. A neglected feature of the p u m p is why internal Na" is not pumped completely out o f cells. Obviously, the control of intracellular ionic composition arises from the influence of internal ions. Emphasis is placed on the activating affect of internal Na ~ and on the competitive inhibition by K +. Competitive inhibition by K ~ brings about regulation o f the p u m p in the following way [84]. As internal Na ~ falls, K + rises and the activation by N a ~ becomes less effective because of competition and a balance is reached beB~.een activation and inhibition. This enables the cells to maintain a constant ionic composition. A further way in which internal K + can regulate the p u m p by inhibiting has been discussed earlier (see Section ILIA-2): this is by direct inhibition arising from the role of K~ as an inhibitory product of the reaction.

I VD. Fhe Simplicity of the Pump Mechanism It is necessary to clarify the general nature of the views expressed here. In the first place, there is no doubt that the phosphoprotein which is sensitive to Na ~, K +

169 and ouabain actually exists and that it is connected with the pump. This material is chemically reactive through enzymic reactions catalyzed by the pump. These reactions, e.g. phosphorylation, can be shown as separate steps, which take place when the normal reaction of the pump is inoperative because not all substrates are present. These reactions demonstrate the catalytic potentialities of the system under conditions where the physiological reaction is, for some reason, blocked. We presented the evidence that they are side reactions, as opposed to partial reactions, branching away from the main flow in the normal, complete pathway. A partial reaction connotes one that is part of the normal overall process and the evidence points against the reactions of the phosphoprotein being of this kind. The phosphoprotein, nevertheless, provides a valuable means of characterising the pump as a chemical entity. The stepwise mechanism (reaction scheme 1) and work on the phosphoprotein is beyond question of the utmost importance in the development of our understanding of the mechanism. However, it will be clear that the experimental discrepancies are so formidable, that we have rejected this mechanism. There are three major conclusions to be drawn from this survey of the mechanism of the sodium pump. First, there are good reasons for the view that ATP hydrolysis and ion movements are linked through a "one-step" reaction. Second, it is likely that the chemical mechanism is fundamentally simple in spite of an impression of bewildering complexity. Third, the branched transition state mechanism provides a neat explanation for the normal and modified modes of operation of the pump which is in line with modern theories of bioenergetics.

V. COMPARISON OF THE PUMP MECHANISM WITH OTHER ENERGY TRANSDUCING SYSTEMS Concepts in energy transformation have undergone a remarkable change as shown when the classical review of Krebs and Kornberg in 1957 [87] is compared with a recent symposium [88]. Earlier views were dominated by the application of thermodynamics to chemical reactions, to the identification of energy donors and acceptors and to the role of ATP as the energy currency [87]. The great contrast in recent discussions stems from the realisation that in the main systems for energy transduction the substrates and products are in compartments separated by membranes in which the necessary enzymes are located. Not only is there heterogeneity, there is also directionality [88]. This vectorial feature is well established for ATP synthesis by tightly coupled mitochondria and chloroplasts. Protons are ejected as ATP is formed and the phosphorylation of A D P depends on the maintenance of proton gradients. It seems likely that in these processes there is no energy donor in the form of a high-energy phosphate chemical intermediate that reacts with ADP. At any rate, none has been convincingly identified and instead it seems that a transition state complex must exist. The flow of electrons connected with proton movements provides the energy for ATP synthesis and the two processes can be regarded as different facets of a single reaction.

170 C o m p a r e this system w i t h t h e t r a n s i t i o n state m e c h a n i s m o f the s o d i u m p u m p we h a v e d e s c r i b e d [39].

T h e r e is v e c t o r i a l flow o f m a t t e r ( N a + a n d K ÷) across a

m e m b r a n e , a n d the p u m p h y d r o l y s e s A T P as the ions are t r a n s p o r t e d w i t h o u t the involvement of chemical intermediates.

It is m i s l e a d i n g to t h i n k o f the t w o p h e n o -

m e n a i.e. t r a n s p o r t a n d c h e m i c a l h y d r o l y s i s as s e p a r a t e : the t w o are i n t e r d e p e n d e n t . It is in the light o f this kind o f b a c k g r o u n d t h a t o u r finding o f s i m u l t a n e o u s t r a n s p o r t o f N a + a n d K + and the a s s o c i a t e d A T P h y d r o l y s i s in a single step r e a c t i o n [55] a c q u i r e s a w i d e r significance.

W h a t e m e r g e s is t h a t the s o d i u m p u m p , as the m a i n

e n e r g y c o n s u m i n g system in t h e b o d y , s h o w s the s a m e f u n d a m e n t a l f e a t u r e s for e n e r g y t r a n s d u c t i o n as d o e s t h e m a i n e n e r g y - y i e l d i n g system, o x i d a t i v e p h o s p h o r y lation. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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