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Zinc: what is its role in biology?
Zinc: what is its role in biology? Robert J. P. Williams Zinc is as common as iron in biology. It is usually thought to be a trace element required only for catalysis. Here it is shown that the role of zinc is much more fundamental not only in catalysis but in inhibitory control. There may well be several such control elements+alcium, iron, manganese, magnesium, and zinc.
It is often thought that the biological role of trace metals is in catalysis. While this article will not deny the fact that this is a large measure of the truth [l] it is far from the whole truth. I shall illustrate here the more general discussion of the role of trace elements with a consideration of the functions of zinc in biology but I believe that these considerations can be extended to iron and manganese biochemistry [2] though possibly not to those of cobalt, nickel, copper, or molybdenum, which may be purely catalytic. In order to appreciate the role of an element such as zinc I shall start the description of zinc activities not from enzymes but from analysis. This approach I learnt from Professor H. M. N. H. Irving and Professor B. L. Vallee to whom I dedicate the article since the three of us have shared at various times a common interestthe determination and location of small quantities of trace elements of unknown function [3]. It will be shown that the location of an element reveals much about its function. The analytical compartments
composition
of zinc
The first point to notice is that zinc is everywhere in biology but it is at very different analytical concentrations. Before descending to cells and then organelles the distribution is most odd even in whole organs. Zinc is very high in the prostate, some parts of some animal eyes, and some parts of the pancreas (Table 1). The liver and some voluntary muscles are also high in zinc. At first sight, these facts tell you nothing but a second look at them
R. J. P. Williams,
M.A., D. Phil., F.R.S.
Was born in 1926 and educated at the Universities of Oxford and Uppsala. He was a Rotary International Fellow in Sweden 1950-51 and since 1974 has been Napier Research Professor of the Royal Society at Oxford. His research interests include complex-ion chemistry and the function of metals in biological systems.
En&avow, New Series, Volume 016&9327/84 SOoO+~50 @ 1984 Pergamon Press.. Printed
8, No. 2,1984. in Great
Britain
forces the train of thought: ‘What is zinc doing in such peculiar places? How did it get there? What for?’ The concentrations in some locations are so high that only two explanations of its concentration are satisfactory. Either the organ produces a very strong binding agent for zinc or it uses energy to pump zinc from the extra-organ fluids. In either case a considerable effort must have gone into the rejection of many other competitive elements, such as iron and manganese. The only way in which we can proceed analytically is to reduce the size of the compartment, looking at the level of the element in the cells and then in the organelles. Once we have discovered the organelle concerned with zinc accumulation it becomes interesting to uncover the associated ligands and/or the pumping mechanism. Cellular
levels
Vallee, who pioneered research into zinc in biology, has told me that he was first intrigued by zinc on noticing that white cells contain a lot and red cells very little. Red cells are full of iron, of course, so zinc and iron are partly separated in different cells even in the blood stream. The iron in the red cell is for oxygen transport. What is the zinc in white cells for? The real question turns out to be why has the red cell lost most of the zinc, since elsewhere many cells have the same high levels of zinc as white cells. The obvious first answer is that zinc is required for many catalysed reactions in normal cells and since red cells have almost stopped metabolism they need no zinc. Unfortunately, two of the best known protein associations of zinc are with insulin and metallothionine which have no catalytic function [l, 41. Proceeding with the analytical inspection it has become clear that zinc enzymes and metallothionine are in most, perhaps all, cells. Further analysis then shows that additional zinc is present in many cells and is distributed amongst a large number of different proteins, often in special vesicles which contain high concentrations in association with particular systems. The over-
all analytical picture is given by Table 1 and figure 1. Now that we have a picture of where zinc is in organisms we must examine the molecules with which it is associated and what it does, starting from its catalytic functions. The potential
catalytic
role of zinc
In principle, zinc can act as a (Lewis) acid polarising, for example, a carbonyl bond or as a conjugate acid required to produce a base and here the natural base for zinc to produce in water is hydroxide, Zn*+-OH-. There is little doubt that zinc fills both roles in one or other of the different enzymes of Table 2. A question must arise as to why biological systems should choose zinc rather than one of the other trace elements such as copper-which is a stronger (Lewis) acid-or iron, which though not such a strong acid is readily available and is common in biological systems generally. The second question is why zinc is used when there are known examples of enzymes which carry out very similar reactions without any trace metal (see below). Zinc does have some special chemical features. The first is its ready formation of low coordination number sites-that is, four of five coordinatewhich are more strongly acidic than high coordination number sites (Table 2 and figure 2). A second feature is the easy deformation of the geometry of the ligands in its coordination sphere, and its ready change of coordination number from four to five to six. It is an advantage in a catalytic site if the catalytic groups have flexibility in their geometric constraints, since the reactants and the products must show mobility through the reaction pathway of several intermediates. A third feature of zinc chemistry is that the ligands in its complex always show relatively rapid exchange. This property has a clear advantage in catalysis. Of course, zinc itself must not be lost from the protein (see below). It is the case, however, that in all these respects it is relatively easy to see how similar properties in catalytic sites could have been devised using either copper (II) or iron 65
MEMBRANE
other reactions catalysed by zinc enzymes, Table 3. One striking exception is the catalysis of the reaction
SIGNALS DIGESTION
ZnP6
VV”““‘~‘V’~““““V~~“‘~
Zn t 5
t1 ZnP,
M ITOCHONDRIA
(ZnMT) -EXCRETE Figure 1 Generalised outline description of the distribution of zinc in cells. ZnP,, protein carrier outside the cell. MT, metallothionine. The vesicle containing MP2 can be for export of enzymes or hormones. Ribosomal, ZnP4, and Nuclear, ZnP3, proteins can be for polymerisation catalysis or protection. ZnP, is, for example, alkaline phosphatase.
(II). (Even iron (III) and manganese (III) are used in acid phosphatases where their functions require the metal to act as an acid. We note, however, that these acid catalysts are found only in acid solutions external to the cell cytoplasm, where zinc proteins would be destroyed. Normally zinc is the preferred Lewis acid). A property peculiar to zinc is the absence of redox chemistry. It could well be, therefore, that many zinc enzymes are used specifically in situations where the presence of redox reactions would lead to damaging radicals and/or preferential reaction with oxygen or hydrogen peroxide. Examples are the polymerisation of RNA and some DNA; the oxidation of alcohols to TABLE 1
Compartment Red blood cell Leucocytes Liver cells Prostate Seminal vesicle Islets of Langerhans Tapetum lucidum
ZINC CONTENT
Proton Bronsted acid catalysts
acid and zinc Lewis
The puzzle of zinc catalysts is why zinc is used at all. There are known enzymes without metals which seemingly do most of the required hydrolyses and
OF BIOLOGICAL
COMPARTMENTS
Total zinc concentration (Moles per litre wet weight) 10-4 10-3 1om3 10-3 10-3 1om2 10
*Estimated from the binding constant insulin, (d) zinc cysteinate at pH7.
66
aldehydes; and perhaps even the hydrolyses of proteins where groups such as tryptophan, tyrosine, and cysteine are readily oxidised. Zinc may well have been selected against copper or iron for just this reason, but this requires deliberate segregation of zinc from iron, copper, and manganese. This case for zinc leaves unanswered the question why and where has zinc been chosen in preference to organic group catalysts?
of (a) methallothionine,
Free [Zn*+l* 4o-6 s1o-6 -IO-lo -1O-5 10m5 -10-5 10m6
(b) zinc citrate,
Here the proton is a poor catalyst. In fact, catalysis appears to require some concerted mechanism using a ‘soft’ base as well as an acid. The chemical catalysts of this reaction are ClOH, Se020H-, SOzOH- (see [5]) and no non-metal, N-, C-, or 0- centred catalyst is very useful. Zinc then acts as Zn*+OH- in a cyclic concerted reaction in this special case. Now CO*, HC03-, and Hz0 are very small substrates and it is also a common feature of redox catalysis in biology that metal ions are used for the most difficult reactions of small molecules where the protein supplies very little in the way of selectivity or strength of binding. Amino-acid side chains are not able to attack small molecules apparently. But what of the many other enzymes in which zinc is used? For factors leading to a possible explanation we must look at the reactions to see if there are any special features (Table 3). We note that zinc is used in terminal amino or carboxy-peptidases with only the occasional exception. Is this an indication of a preference for zinc when the specificity of binding on one or both sides of the bond to be broken is not wanted? Zinc is used in the handling of general phosphate hydrolyses (phosphatase) or general nucleotide phosphate ester formation (some DNA and RNA) but not in specified phosphate transfer. Could it be that at a site of low selectivity organic sidechains are not able to generate good catalysis but that even in this circumstance zinc can generate good water or hydroxide ion catalysis? The suggestion is that metals such as zinc are much stronger functional reagents than the side chains of proteins. Table 3 lists which enzymes use zinc, some which do not, and some where overlap between the two types of enzymes occurs. There is general support for the suggestion. It then seems highly probable that enzymes which do not use metal ions depend upon strain due to binding rather than upon good attacking groups. Naturally, this gives greater selectivity but is only a possible form of attack with larger substrates. The entatic
(a) (b) (b) (c) (d)
(c) zinc
state of zinc
In order to make optimum catalysis from a given group, organic or inorganic, its reactivity should be enhanced relative to that of the simple isolated group. Zinc in conventional complex ions is not a very good acid. The fold of proteins can generate not only special ligands but also special stereochemistries so as to heighten.activity. In other words, the protein fold and its
TABLE 2
SOME ZINC ENZYMES
Enzyme
Structural
Carbonic anhydrase Carboxypeptidases Alkaline phosphatase
4/5 5/6 4/5 5/6 4/5
Alcohol
dehydrogenase
(a) (b)
features
Coordinate Coordinate Coordinate Coordinate? Coordinate
binding and kinetic selection of a pathway for the element to the site. Let us suppose that a protein A is produced in the cytoplasm of a cell. The metal which binds to A is selected in part by the chemistry of the side-chains of A in a particular fold (we will return to the fold energy later). For the moment, we know that some proteins can offer amine, thiolate, carboxylate, and so on binding groups. There is an immediate discrimination in favour of zinc and the transition metals and against magnesium and calcium if the fold places together two or more nitrogen or sulphur bases. Moreover the discrimination is in the Irving-Williams order of divalent ions
111 (active
site)
3N. (H20)1-2 2N. -COz-, o-&,0),-, 3N? (HzO),-z N, (-CO*-), Hz0 N, 2RS, (HzO),-,
Enzymes of unknown structure include Aldolase, B-Lactamase, Saccharases, Polymerases (DNA and RNA), Glyoxalase, see A. Galdes and B. L. Vallee in Ref. (11
energy states make zinc more active and more readily able to go along a required reaction path than would be true in less sophisticated complexes. This pre-setting of the state of a group by a protein fold is called the entatic state. As yet we can only note the very different coordination geometries of zinc in different enzymes, indicating that some such pre-setting occurs (figure 2), but we have no well-defined notions about the correlation between particular zinc sites and their enzymic activities. Zinc structural
so that limited relaxation of the copper coordination sphere during the oxidation state change (Cu(I)--,Cu(II) is permitted. This then is a special case of the ‘entatic’ ground state of copper, with a special mobility constraint added to the excited states. Some relaxation modes of bonds are required in reactions but if they are to be fast there must not be too many modes.
Cu>Zn>Ni>Co>Fe>Mn>Mg>Ca The increments between elements in this order are quite large, so that if there were an excess availability of all elements Cu(I1) would take all proteins including protein A. Clearly, there
roles
Zinc has a structural role in certain intracellular proteins. It cross-links the proteins in Table 4. In a sense, it takes the place of a disulphide bridge forming a knot in the chain of the protein. It has two advantages over the disulphide bridge. First, it cannot be reduced so that it is stable even in the reducing atmosphere of the inside of cells. Second, since zinc has little stereochemical demand it allows some motion about itself while it restricts the conformational modes very considerably. We may then suppose, for example, that one zinc in alcohol dehydrogenase is used in this way. Zinc in superoxide dismutase helps to stabilise the unusual geometry around the catalytic copper centre but perhaps it also allows sufficient mobility to the centre
TABLE 3 Reaction
Hydration Protein hydrolysis
spheres of some known zinc sites in enzymes. Left, Figure 2 The coordination carboxypeptidases and probably other proteases; centre, 3N ligands, carbonic anhydrase, and probably aldolase and phosphatase; centre IN and 2s ligands, alcohol dehydrogenase active site; right, alcohol dehydrogenase (structure site) and in part metallothionine.
must be some other discrimination. As in analytical practice the presence of copper must be masked if zinc is to bind to A. A simple procedure is to add a second reagent B with an increment of affinity between copper and zinc which exceeds that which A generates. Copper then takes B first. However, zinc will not combine with A unless the combination of copper with
Before turning away from zinc enzymes we need to look at the actual sites of binding in order to see how zinc and not other metals comes to be bound. The selection ligands
of zinc
by
protein
The uptake of an element into a specific site is a product of thermodynamic
COMPARISON
BETWEEN ZINC AND NON-ZINC
Zinc-dependent enzyme substrate
ENZYMES
Metal-independent substrate None Endopeptide
Phosphate ester hydrolysis RNA/DNA hydrolysis RNAI(DNA) polymerase
COz, levalinic acid Carboxy-terminal, amino terminal and di-peptides Terminal phosphate Non-specified nucleotides No specific bases
R-lactamase* Aldolase’ Phospholipase*
Penicillins Several aldols (C) Lipid esters
Penicillins Several aldols (A) Lipid Esters
enzyme
Phosphate transfer Ribonucleotides None
Note: Generally zinc is employed when the substrate is small, e.g. CO *, or when the reaction is non-specific, e.g. alkaline phosphatase, RNA polymerase, terminal proteases. *Indicates enzymes which are known in two forms, with and without zinc.
67
TABLE 4
STRUCTURAL
ZINC IN PROTEINS Character
Exchange
Proteins
None None None None Exchangeable Fast Fast
Alcohol dehydrogenase Aspartate transcarbamylase Superoxide dismutase Keratin (hair) Keratin (sperm) Insulin [Metallothionine
(?I
ing free-ion levels is part of what is called hoemeostasis and, in outline, in real cells it works as follows. Zinc (and copper) enter cells by controlled methods using proteins (figures 1 and 3). A major binding reagent (B) in the cell is metallothionine which binds copper and zinc very tightly through many thiolate groups [4]. The binding of copper to metallothionine greatly exceeds that of zinc with the result that of all centres A available in cells only those which are very powerful can obtain copper. The few proteins which bind copper in cells all contain four-coordinate N- or Sbinding copper (II) sites, superoxide dismutase and blue electron transfer proteins. All the rest of the copper is bound to metallothionine (B) largely as Cu(1) and as the concentration of this CUB masking agent grows so it and copper are pumped out into extracellular compartments. As a consequence,
B so reduces the copper concentration that the zinc reaction can occur. A biological cell has two possibilities open to it. It can bias the entry to the cell of zinc and copper in favour of zinc but note that this means that it must find a way not based on thermodynamics by which a third reagent C carries zinc into a cell in preference to copper. This transfer cannot be based on binding strengths, thermodynamics, alone since that of copper is always greater than that of zinc. Alternatively, the cell can actively reject copper by making an excess of protein B over copper and then pumping the complex CUB out of the cell leaving only a low free copper. This compares with solvent extraction using selective organic reagents in separations in analysis. Biology then makes both a passive thermodynamic selection and an active energy-using selection to develop control over the free-ion levels. This mode of controll-
Lur2 /
I
CuMT +
EXCRETE
Figure 3 Distribution pattern of copper in cells (cf. figure 1. MT, metallothionine. The only known copper proteins in cells are associated with mitochondrial or chloroplast membranes, apart from cytoplasmic Cu/Zn superoxide dismutase. There are many copper oxidases outside the cell or in vesicles for export.
68
of zinc binding
Zn(RS) tetrahedral Zn(RS-) tetrahedral Zn(N) tetrahedral Zn(RS) coordination Zn(RS-) coordination Zn(N), octahedral Zn(RS) tetrahedral]
there are many extra cellular copper enzymes-for example oxidases-and very few in cells (figure 3). The cell is left with a relatively high zinc (compared to copper) concentration, which now faces competition only from Mn(II), Fe(II), and Ni(I1). We note that zinc is favoured by simple thermodynamic considerations especially for tetrahedral protein sites since the stability order for such sites is Zn>Ni>Fe>Mn. (We have omitted cobalt here since it is not readily available due to its low abundance). The problem now is, of course, that zinc has been lowered in its free concentration by combination with excess metallothionine (B) to a much greater degree than has the concentration of the other cations so, in concentration terms, we expect [Mn(II)]>[Fe(II)]>[Ni(II)]>[Zn(II)] New kinetic and thermodynamic devices are needed to correct this bias if zinc is to be selectively taken into proteins, A. Mn(I1) is larger than the other divalent cations and is selectively pumped from cells by the outward Ca(I1) pumps. It goes into vesicles where it is used. This pump does not pump ions of the same size as Mg(II), that is Ni(I1) or Zn(I1). Next we note that Ni(I1) is not very available and is removed preferentially by octahedral not tetrahedral sites. This is a ligandfield selection and takes nickel into urease, for example. Finally iron isdealt with by two devices [2]. Much iron combines with such ligands as citrate in the cell, becoming Fe(II1) citrate. This iron citrate is pumped from the cytoplasm into mitochondria. The input of iron from outside the cell is also controlled by a special protein, transferring (D), and inside the cell by a homeostatic protein, ferritin (a second kind of B protein especially designed for controlling trivalent ions). Thus Fe(I1) is also very low in the cytoplasm due to its oxidation to Fe(II1) and its subsequent removal via ferritin to mitochondria or chloroplasts-that is Fe(II)-is governed by Fe(II1) concentration. The cytoplasm has now a regulated concentration of all the metal ions and can provide suitably selective proteins A for the uptake of zinc,
Before leaving this point, notice that different compartments are biased toward different elements. The cytoplasm is dominated by magnesium and zinc, the mitochondria by iron, different vesicles by calcium and manganese (and, as we shall see in the case of a few vesicles, by zinc), and the extracellular fluids by calcium, copper, and molybdenum. The procedure is very like that of the analytical separation of elements by solvent extraction and/or precipitation followed by the transfer of separated phases. At each membrane selective pumping directs elements into selected compartments. The fold energy of the proteins which capture the metals are now seen to be used in several ways: (1) To generate thermodynamic selectivity. (2) To control exchange rates (see below). (3) To heighten catalytic significance, the entatic state. (4) To undergo conformational switches required, for example, in cation pumps-that is, to have two states of different binding strengths. (5) To permit recognition at membrane surfaces. The
selectivity
of metaliothionein
PI Metallothionein is not a usual protein. It is highly basic, has plenty of thiolate groups and no aromatic residues. It does not fold in the absence of metal ions. When bound to metal it folds. This means that the precise fold is not decided by the sequence but by the metal ions which it binds. The major metals associated with it in biological systems are zinc, cadmium, and copper. The fold with zinc is unlikely to be very similar to that when copper is bound, since copper (I) is much larger and copper (II) demands a very different stereochemistry. Receptor sites on membranes can bind the stereochemitally different metal-protein complexes selectively when it is possible that copper can be eliminated from the cytoplasm while zinc is retained to a large degree. This is the opposite use of a protein to that of transferrin, which very Aelectively introduces iron into cells and can also carry manganese. Metallothionein then has two functions, homeostasis of copper and zinc at very different cytoplasmic levels. (It also aids the removal or elimination of cadmium). Finally, in the folded form it binds to either RNA or DNA, through its basic amino acid side chains, leading to feed-back control over the production of the protein. The fact that metallothionine has a low fold-energy but a high binding constant, while the zinc enzymes have high fold energies and high binding
constants, is a consequence of their different protein sequences. Apart from binding energy differences these different fold energies control exchange rates, since on its own zinc exchanges relatively rapidly from inorganic complexes. The control by different proteins over exchange is undoubtedly a major feature of zinc biochemistry which we will examine below. Reversible
and irreversible
binding
All the known zinc enzymes can be isolated virtually without loss of zinc. This means that in the cell cytoplasm zinc enzymes are not able to exchange zinc freely and cannot be regulated by zinc exchange, although they can be specifically inhibited by zinc-binding reagents. Zinc exchange from metallothionine is relatively fast, however, so that zinc metallothionine can be a part of a regulation system but it is not yet clear what zinc functions it regulates. Clearly, it cannot directly affect the many irreversibly formed zinc enzymes which are produced in vesiclesfor example carboxypeptidase or the irreversibly formed cytoplasmic enzymes-unless there is rapid protein turn-over, which is not known. A clue to the real value of zinc regulationthat is of metallothionine-is to be found in the requirements for zinc in vesicles (Table l), where it is in very high concentration and can be exchanged readily. Zinc in vesicles
We have now seen how zinc enzymes can be preferentially formed in the cell cytoplasm but zinc proteins are also formed in special vesicles-for example, the islets of Langehans (insulin) Pollen
Sperm
Figure 4 Diagrammatic picture of the heavy concentration of zinc in gametes. ZnPl, an inhibitor enzyme, acrosin, in the vesicles of the sperm head; ZnP,, a keratin-like tail. Zinc in the tube of pollen may well be to protect RNA, while in the nuclear region it protects DNA.
and the vesicles of the pancreas which contain digestive zinc enzymes (peptidases, amylases, and lipases). The movement of protein through the Golgi membrane to the interior of these vesicles needs an unfolded protein which cannot carry zinc. It follows that some Golgi vesicles (which become the islets of Langehans or digestive vesicles) must specifically pump in zinc. This is possible, of course, once the copper has been sufficiently removed from the cytoplasm by metallothionine. Now the free zinc concentration in some of these well-known vesicles must be high (alO-*M) relative to that established by metallothionine in the cytoplasm (lo-‘*M) since the binding constants of zinc to insulin or cysteine, for example, at pH7 are low (Table 1). What do we know about such vesicles? For the most part, the answer is very little but knowledge is accumulating about the very special cases of the reproductive gametes and associated vesicle systems (figure 4) 17-121. There appear to be two different zinc fractions in both sperm and pollen [8, 121. One, the larger, is associated in sperm with the tail of the male gamete, which is made of keratin. As far as can be judged the function of the zinc here is to bind to sulphydryl groups partly cross-linking the keratin and helping to avoid oxidative damage to it. This is very similar to the funciton of zinc in hair and nails. This part of the zinc is really extracellular, since the sperm are held in a fluid space which is to be ejected and the tail is air extension of the male cell outside its cytoplasmic membrane. (There is some similarity with the insulin vesicles). The zinc in the head of the sperm cells is intracellular and some is probably in a special vesicular system said to be associated with m-RNA and/or a lysozomal vesicle. The situation is clearer in pollen [12]. The developed pollen cell has two zinc regions: a long tube through which the male DNA will pass and a head which retains the DNA. There is at the tip of the tube a high concentration of zinc in association with a vesicular recticulum which also carries messenger RNA. The idea that zinc actually binds this RNA is improbable, since it is known that all RNA has very poor zinc-binding capability. It is more likely that the m-RNA is associated with a protein which protects the RNA and prevents transcription. A reasonable guess would be that this is a thiol-containing protein to bind zinc and that the zinc protein folds so as to bind the m-RNA when the protein becomes similar to metallothionines which have a very positive surface (compare histones) and they do bind to DNA. Such proteins could protect RNA and DNA as is required 69
TABLE 5
SOME SYSTEMS
Process
Molecular
Nerve growth factor Keratin-like sperm tail* Sperm protease (acrosin) DNA & RNA condensations Spore germination Prohormone hydrolysis?
Zinc Zinc Zinc Zinc Zinc Zinc
*Keratin
filaments
BY ZINC
characteristics
prevents protects inhibits proteins release controls
111 El
enzyme activation from break-down activation stabilise very early enzymes?
171
P31 191 111
may also have a major role inside cells and even in cell division.
in condensed genetic material in gametes. There is also much zinc in association with the DNA in the head of the pollen. Returning to the mammalian reproductive system, it is known that during sperm ejaculation the sperm is surrounded by a fluid from the prostrate which is very rich in zinc and citrate compounds [ 10-111. The zinc/citrate solution seemingly adds further protection to the sperm, especially the tail, maintaining as long as possible the zinc and a reducing environment. This environment is (slowly) lost in the female reproductive tract, where the zinc is in relatively fast exchange and low concentration. The zinc may well be associated with citrate in vesicles before ejection. The zinc is in fast exchange, since zinc citrate is a relatively weak complex [ll]. Another example of zinc in fast exchange is the zinc cysteinate of the vesicles of the tapetum lucidem of the eye. Here zinc must be in fast exchange since zinc cysteinate is not so very stable at pH7. The free zinc is probably lo-‘M. Another parallel is to be found in the venom-containing vesicles of snakes which contain NGF, nerve growth factors (enzymes), in a protected state. In some cases it has been demonstrated that the zinc of NGF acts to stabilise the NGF precursor zymogen. Relatively easy loss of zinc liberates the NGF-enzyme but addition of zinc inhibits the conversion of the pro-enzyme. NGF, like the zinc cysteinate, is stored in a vesicle. These several observations on vesicle or specially protected cell systems (Table 5) lead to a new hypothesis for a major role of zinc. It is a protective/ trigger agent in pumped storage sys-
70
INHIBITED
tems in association with small chelates or proteins often containing thiols. It is a trigger in that it prevents reactions of DNA, RNA, or proteins (Table 5) until they are required. The zinc trigger is then the local drop of zinc concentration after release from the vesicle. By controlling enzymes, zinc can stabilise zymogens (nerve growth factor), and hormones (insulin) through stabilising prohormones and/or through polymerisation. Zinc pro-insulin is then a zinc buffer to prevent insulin activation in two ways. Zinc may also control the folds of RNA or DNA in inhibited forms in association with thiol-binding groups of amino acids in vesicles. The homeostasis of zinc in cell cytoplasm is then to be associated with the pumping of the zinc to high levels in many special vesicular systems. By contrast, the zinc in the normal cell cytoplasm is in a separate kinetic compartment where it is largely in irreversible complexes and required for fast catalysis of reactions of low specificity. Comparison with calcium is instructive. Free calcium is the biological trigger of fast reactions and its binding to proteins gives activation either in the cell or outside it. Free calcium ions are stored. In the case of zinc, bound zinc complexes are stored and activity is triggered by dilution which releases the reagents from zinc. If this is true we must search for zinc-containing vesicles in many cells. It may well have a special relationship with cell-growth processes, for example in reproduction and in brain [13], and together with calcium it may provide a very general set of control reactions. Finally, zinc may associate with keratin-like filaments both inside and outside cells.
References
[l] Sigel, H (ed.) ‘Zinc and Its Role in Biology and Nutrition’, Vol. 15 of ‘Metal Ions in Biological Systems’, Dekker, New York, 1983. [2] Williams, R. J. P. FEBS Letters, 703, 87, 1982. [3] Vallee, B. L. and Falchuk, K. Phil Trans. Roy. Sot. London, 294B, 185, 1981.
[4] Kagi, J. H. T. and Nordberg, M. ‘Metallothionine’, Birkhauser Verlag, Base], 1981. [5] Dennard, A. E. and Williams, R. J. P., J. Chem. Sot., (London), 1966A, 612 [6] Vallee, B. L. and Williams, R. J. P. Proc. Natl. Acad.
Sci. (U.S.),
59,
498, 1968. [7] Steven, F. S., Griffin, M. M. and Chantler, E. N. Int. J. Andrology, 5, 401, 1982.
[8] Roomans, G. M., Lundevall, E., Bjorndahl, L., and Krist, U. Znternut. J. Andrology, 5, 478, 1982. [9] Johnston, K., Stewart, G. S. A. B., Scott, I. R., and Ellar, D. J. Biochem. J., 208, 407, 1982.
[lo] Mann, T. and Lutwak-Mann, C., ‘Male Reproductive Function and Semen’, SpringerVerlag, Berlin 1981. [ll] Arver, S., Acta Physiol. Stand. 116, 67, 1982. [12] Ender, C. H., Li, M. Q., Martin B., Park B., Nobilung R., ReissH-D., and Traxel K. Protoplasm 1983, submitted. [13] Itoh, M., Ebadi, M., and Swanson,S., J. Neurochemistry, 41, 823, 1983.
It is often thought that the biological role of trace metals is in catalysis. While this article will not deny the fact that this is a large measure of the truth [l] it is far from the whole truth. I shall illustrate here the more general discussion of the role of trace elements with a consideration of the functions of zinc in biology but I believe that these considerations can be extended to iron and manganese biochemistry [2] though possibly not to those of cobalt, nickel, copper, or molybdenum, which may be purely catalytic. In order to appreciate the role of an element such as zinc I shall start the description of zinc activities not from enzymes but from analysis. This approach I learnt from Professor H. M. N. H. Irving and Professor B. L. Vallee to whom I dedicate the article since the three of us have shared at various times a common interestthe determination and location of small quantities of trace elements of unknown function [3]. It will be shown that the location of an element reveals much about its function. The analytical compartments
composition
of zinc
The first point to notice is that zinc is everywhere in biology but it is at very different analytical concentrations. Before descending to cells and then organelles the distribution is most odd even in whole organs. Zinc is very high in the prostate, some parts of some animal eyes, and some parts of the pancreas (Table 1). The liver and some voluntary muscles are also high in zinc. At first sight, these facts tell you nothing but a second look at them
R. J. P. Williams,
M.A., D. Phil., F.R.S.
Was born in 1926 and educated at the Universities of Oxford and Uppsala. He was a Rotary International Fellow in Sweden 1950-51 and since 1974 has been Napier Research Professor of the Royal Society at Oxford. His research interests include complex-ion chemistry and the function of metals in biological systems.
En&avow, New Series, Volume 016&9327/84 SOoO+~50 @ 1984 Pergamon Press.. Printed
8, No. 2,1984. in Great
Britain
forces the train of thought: ‘What is zinc doing in such peculiar places? How did it get there? What for?’ The concentrations in some locations are so high that only two explanations of its concentration are satisfactory. Either the organ produces a very strong binding agent for zinc or it uses energy to pump zinc from the extra-organ fluids. In either case a considerable effort must have gone into the rejection of many other competitive elements, such as iron and manganese. The only way in which we can proceed analytically is to reduce the size of the compartment, looking at the level of the element in the cells and then in the organelles. Once we have discovered the organelle concerned with zinc accumulation it becomes interesting to uncover the associated ligands and/or the pumping mechanism. Cellular
levels
Vallee, who pioneered research into zinc in biology, has told me that he was first intrigued by zinc on noticing that white cells contain a lot and red cells very little. Red cells are full of iron, of course, so zinc and iron are partly separated in different cells even in the blood stream. The iron in the red cell is for oxygen transport. What is the zinc in white cells for? The real question turns out to be why has the red cell lost most of the zinc, since elsewhere many cells have the same high levels of zinc as white cells. The obvious first answer is that zinc is required for many catalysed reactions in normal cells and since red cells have almost stopped metabolism they need no zinc. Unfortunately, two of the best known protein associations of zinc are with insulin and metallothionine which have no catalytic function [l, 41. Proceeding with the analytical inspection it has become clear that zinc enzymes and metallothionine are in most, perhaps all, cells. Further analysis then shows that additional zinc is present in many cells and is distributed amongst a large number of different proteins, often in special vesicles which contain high concentrations in association with particular systems. The over-
all analytical picture is given by Table 1 and figure 1. Now that we have a picture of where zinc is in organisms we must examine the molecules with which it is associated and what it does, starting from its catalytic functions. The potential
catalytic
role of zinc
In principle, zinc can act as a (Lewis) acid polarising, for example, a carbonyl bond or as a conjugate acid required to produce a base and here the natural base for zinc to produce in water is hydroxide, Zn*+-OH-. There is little doubt that zinc fills both roles in one or other of the different enzymes of Table 2. A question must arise as to why biological systems should choose zinc rather than one of the other trace elements such as copper-which is a stronger (Lewis) acid-or iron, which though not such a strong acid is readily available and is common in biological systems generally. The second question is why zinc is used when there are known examples of enzymes which carry out very similar reactions without any trace metal (see below). Zinc does have some special chemical features. The first is its ready formation of low coordination number sites-that is, four of five coordinatewhich are more strongly acidic than high coordination number sites (Table 2 and figure 2). A second feature is the easy deformation of the geometry of the ligands in its coordination sphere, and its ready change of coordination number from four to five to six. It is an advantage in a catalytic site if the catalytic groups have flexibility in their geometric constraints, since the reactants and the products must show mobility through the reaction pathway of several intermediates. A third feature of zinc chemistry is that the ligands in its complex always show relatively rapid exchange. This property has a clear advantage in catalysis. Of course, zinc itself must not be lost from the protein (see below). It is the case, however, that in all these respects it is relatively easy to see how similar properties in catalytic sites could have been devised using either copper (II) or iron 65
MEMBRANE
other reactions catalysed by zinc enzymes, Table 3. One striking exception is the catalysis of the reaction
SIGNALS DIGESTION
ZnP6
VV”““‘~‘V’~““““V~~“‘~
Zn t 5
t1 ZnP,
M ITOCHONDRIA
(ZnMT) -EXCRETE Figure 1 Generalised outline description of the distribution of zinc in cells. ZnP,, protein carrier outside the cell. MT, metallothionine. The vesicle containing MP2 can be for export of enzymes or hormones. Ribosomal, ZnP4, and Nuclear, ZnP3, proteins can be for polymerisation catalysis or protection. ZnP, is, for example, alkaline phosphatase.
(II). (Even iron (III) and manganese (III) are used in acid phosphatases where their functions require the metal to act as an acid. We note, however, that these acid catalysts are found only in acid solutions external to the cell cytoplasm, where zinc proteins would be destroyed. Normally zinc is the preferred Lewis acid). A property peculiar to zinc is the absence of redox chemistry. It could well be, therefore, that many zinc enzymes are used specifically in situations where the presence of redox reactions would lead to damaging radicals and/or preferential reaction with oxygen or hydrogen peroxide. Examples are the polymerisation of RNA and some DNA; the oxidation of alcohols to TABLE 1
Compartment Red blood cell Leucocytes Liver cells Prostate Seminal vesicle Islets of Langerhans Tapetum lucidum
ZINC CONTENT
Proton Bronsted acid catalysts
acid and zinc Lewis
The puzzle of zinc catalysts is why zinc is used at all. There are known enzymes without metals which seemingly do most of the required hydrolyses and
OF BIOLOGICAL
COMPARTMENTS
Total zinc concentration (Moles per litre wet weight) 10-4 10-3 1om3 10-3 10-3 1om2 10
*Estimated from the binding constant insulin, (d) zinc cysteinate at pH7.
66
aldehydes; and perhaps even the hydrolyses of proteins where groups such as tryptophan, tyrosine, and cysteine are readily oxidised. Zinc may well have been selected against copper or iron for just this reason, but this requires deliberate segregation of zinc from iron, copper, and manganese. This case for zinc leaves unanswered the question why and where has zinc been chosen in preference to organic group catalysts?
of (a) methallothionine,
Free [Zn*+l* 4o-6 s1o-6 -IO-lo -1O-5 10m5 -10-5 10m6
(b) zinc citrate,
Here the proton is a poor catalyst. In fact, catalysis appears to require some concerted mechanism using a ‘soft’ base as well as an acid. The chemical catalysts of this reaction are ClOH, Se020H-, SOzOH- (see [5]) and no non-metal, N-, C-, or 0- centred catalyst is very useful. Zinc then acts as Zn*+OH- in a cyclic concerted reaction in this special case. Now CO*, HC03-, and Hz0 are very small substrates and it is also a common feature of redox catalysis in biology that metal ions are used for the most difficult reactions of small molecules where the protein supplies very little in the way of selectivity or strength of binding. Amino-acid side chains are not able to attack small molecules apparently. But what of the many other enzymes in which zinc is used? For factors leading to a possible explanation we must look at the reactions to see if there are any special features (Table 3). We note that zinc is used in terminal amino or carboxy-peptidases with only the occasional exception. Is this an indication of a preference for zinc when the specificity of binding on one or both sides of the bond to be broken is not wanted? Zinc is used in the handling of general phosphate hydrolyses (phosphatase) or general nucleotide phosphate ester formation (some DNA and RNA) but not in specified phosphate transfer. Could it be that at a site of low selectivity organic sidechains are not able to generate good catalysis but that even in this circumstance zinc can generate good water or hydroxide ion catalysis? The suggestion is that metals such as zinc are much stronger functional reagents than the side chains of proteins. Table 3 lists which enzymes use zinc, some which do not, and some where overlap between the two types of enzymes occurs. There is general support for the suggestion. It then seems highly probable that enzymes which do not use metal ions depend upon strain due to binding rather than upon good attacking groups. Naturally, this gives greater selectivity but is only a possible form of attack with larger substrates. The entatic
(a) (b) (b) (c) (d)
(c) zinc
state of zinc
In order to make optimum catalysis from a given group, organic or inorganic, its reactivity should be enhanced relative to that of the simple isolated group. Zinc in conventional complex ions is not a very good acid. The fold of proteins can generate not only special ligands but also special stereochemistries so as to heighten.activity. In other words, the protein fold and its
TABLE 2
SOME ZINC ENZYMES
Enzyme
Structural
Carbonic anhydrase Carboxypeptidases Alkaline phosphatase
4/5 5/6 4/5 5/6 4/5
Alcohol
dehydrogenase
(a) (b)
features
Coordinate Coordinate Coordinate Coordinate? Coordinate
binding and kinetic selection of a pathway for the element to the site. Let us suppose that a protein A is produced in the cytoplasm of a cell. The metal which binds to A is selected in part by the chemistry of the side-chains of A in a particular fold (we will return to the fold energy later). For the moment, we know that some proteins can offer amine, thiolate, carboxylate, and so on binding groups. There is an immediate discrimination in favour of zinc and the transition metals and against magnesium and calcium if the fold places together two or more nitrogen or sulphur bases. Moreover the discrimination is in the Irving-Williams order of divalent ions
111 (active
site)
3N. (H20)1-2 2N. -COz-, o-&,0),-, 3N? (HzO),-z N, (-CO*-), Hz0 N, 2RS, (HzO),-,
Enzymes of unknown structure include Aldolase, B-Lactamase, Saccharases, Polymerases (DNA and RNA), Glyoxalase, see A. Galdes and B. L. Vallee in Ref. (11
energy states make zinc more active and more readily able to go along a required reaction path than would be true in less sophisticated complexes. This pre-setting of the state of a group by a protein fold is called the entatic state. As yet we can only note the very different coordination geometries of zinc in different enzymes, indicating that some such pre-setting occurs (figure 2), but we have no well-defined notions about the correlation between particular zinc sites and their enzymic activities. Zinc structural
so that limited relaxation of the copper coordination sphere during the oxidation state change (Cu(I)--,Cu(II) is permitted. This then is a special case of the ‘entatic’ ground state of copper, with a special mobility constraint added to the excited states. Some relaxation modes of bonds are required in reactions but if they are to be fast there must not be too many modes.
Cu>Zn>Ni>Co>Fe>Mn>Mg>Ca The increments between elements in this order are quite large, so that if there were an excess availability of all elements Cu(I1) would take all proteins including protein A. Clearly, there
roles
Zinc has a structural role in certain intracellular proteins. It cross-links the proteins in Table 4. In a sense, it takes the place of a disulphide bridge forming a knot in the chain of the protein. It has two advantages over the disulphide bridge. First, it cannot be reduced so that it is stable even in the reducing atmosphere of the inside of cells. Second, since zinc has little stereochemical demand it allows some motion about itself while it restricts the conformational modes very considerably. We may then suppose, for example, that one zinc in alcohol dehydrogenase is used in this way. Zinc in superoxide dismutase helps to stabilise the unusual geometry around the catalytic copper centre but perhaps it also allows sufficient mobility to the centre
TABLE 3 Reaction
Hydration Protein hydrolysis
spheres of some known zinc sites in enzymes. Left, Figure 2 The coordination carboxypeptidases and probably other proteases; centre, 3N ligands, carbonic anhydrase, and probably aldolase and phosphatase; centre IN and 2s ligands, alcohol dehydrogenase active site; right, alcohol dehydrogenase (structure site) and in part metallothionine.
must be some other discrimination. As in analytical practice the presence of copper must be masked if zinc is to bind to A. A simple procedure is to add a second reagent B with an increment of affinity between copper and zinc which exceeds that which A generates. Copper then takes B first. However, zinc will not combine with A unless the combination of copper with
Before turning away from zinc enzymes we need to look at the actual sites of binding in order to see how zinc and not other metals comes to be bound. The selection ligands
of zinc
by
protein
The uptake of an element into a specific site is a product of thermodynamic
COMPARISON
BETWEEN ZINC AND NON-ZINC
Zinc-dependent enzyme substrate
ENZYMES
Metal-independent substrate None Endopeptide
Phosphate ester hydrolysis RNA/DNA hydrolysis RNAI(DNA) polymerase
COz, levalinic acid Carboxy-terminal, amino terminal and di-peptides Terminal phosphate Non-specified nucleotides No specific bases
R-lactamase* Aldolase’ Phospholipase*
Penicillins Several aldols (C) Lipid esters
Penicillins Several aldols (A) Lipid Esters
enzyme
Phosphate transfer Ribonucleotides None
Note: Generally zinc is employed when the substrate is small, e.g. CO *, or when the reaction is non-specific, e.g. alkaline phosphatase, RNA polymerase, terminal proteases. *Indicates enzymes which are known in two forms, with and without zinc.
67
TABLE 4
STRUCTURAL
ZINC IN PROTEINS Character
Exchange
Proteins
None None None None Exchangeable Fast Fast
Alcohol dehydrogenase Aspartate transcarbamylase Superoxide dismutase Keratin (hair) Keratin (sperm) Insulin [Metallothionine
(?I
ing free-ion levels is part of what is called hoemeostasis and, in outline, in real cells it works as follows. Zinc (and copper) enter cells by controlled methods using proteins (figures 1 and 3). A major binding reagent (B) in the cell is metallothionine which binds copper and zinc very tightly through many thiolate groups [4]. The binding of copper to metallothionine greatly exceeds that of zinc with the result that of all centres A available in cells only those which are very powerful can obtain copper. The few proteins which bind copper in cells all contain four-coordinate N- or Sbinding copper (II) sites, superoxide dismutase and blue electron transfer proteins. All the rest of the copper is bound to metallothionine (B) largely as Cu(1) and as the concentration of this CUB masking agent grows so it and copper are pumped out into extracellular compartments. As a consequence,
B so reduces the copper concentration that the zinc reaction can occur. A biological cell has two possibilities open to it. It can bias the entry to the cell of zinc and copper in favour of zinc but note that this means that it must find a way not based on thermodynamics by which a third reagent C carries zinc into a cell in preference to copper. This transfer cannot be based on binding strengths, thermodynamics, alone since that of copper is always greater than that of zinc. Alternatively, the cell can actively reject copper by making an excess of protein B over copper and then pumping the complex CUB out of the cell leaving only a low free copper. This compares with solvent extraction using selective organic reagents in separations in analysis. Biology then makes both a passive thermodynamic selection and an active energy-using selection to develop control over the free-ion levels. This mode of controll-
Lur2 /
I
CuMT +
EXCRETE
Figure 3 Distribution pattern of copper in cells (cf. figure 1. MT, metallothionine. The only known copper proteins in cells are associated with mitochondrial or chloroplast membranes, apart from cytoplasmic Cu/Zn superoxide dismutase. There are many copper oxidases outside the cell or in vesicles for export.
68
of zinc binding
Zn(RS) tetrahedral Zn(RS-) tetrahedral Zn(N) tetrahedral Zn(RS) coordination Zn(RS-) coordination Zn(N), octahedral Zn(RS) tetrahedral]
there are many extra cellular copper enzymes-for example oxidases-and very few in cells (figure 3). The cell is left with a relatively high zinc (compared to copper) concentration, which now faces competition only from Mn(II), Fe(II), and Ni(I1). We note that zinc is favoured by simple thermodynamic considerations especially for tetrahedral protein sites since the stability order for such sites is Zn>Ni>Fe>Mn. (We have omitted cobalt here since it is not readily available due to its low abundance). The problem now is, of course, that zinc has been lowered in its free concentration by combination with excess metallothionine (B) to a much greater degree than has the concentration of the other cations so, in concentration terms, we expect [Mn(II)]>[Fe(II)]>[Ni(II)]>[Zn(II)] New kinetic and thermodynamic devices are needed to correct this bias if zinc is to be selectively taken into proteins, A. Mn(I1) is larger than the other divalent cations and is selectively pumped from cells by the outward Ca(I1) pumps. It goes into vesicles where it is used. This pump does not pump ions of the same size as Mg(II), that is Ni(I1) or Zn(I1). Next we note that Ni(I1) is not very available and is removed preferentially by octahedral not tetrahedral sites. This is a ligandfield selection and takes nickel into urease, for example. Finally iron isdealt with by two devices [2]. Much iron combines with such ligands as citrate in the cell, becoming Fe(II1) citrate. This iron citrate is pumped from the cytoplasm into mitochondria. The input of iron from outside the cell is also controlled by a special protein, transferring (D), and inside the cell by a homeostatic protein, ferritin (a second kind of B protein especially designed for controlling trivalent ions). Thus Fe(I1) is also very low in the cytoplasm due to its oxidation to Fe(II1) and its subsequent removal via ferritin to mitochondria or chloroplasts-that is Fe(II)-is governed by Fe(II1) concentration. The cytoplasm has now a regulated concentration of all the metal ions and can provide suitably selective proteins A for the uptake of zinc,
Before leaving this point, notice that different compartments are biased toward different elements. The cytoplasm is dominated by magnesium and zinc, the mitochondria by iron, different vesicles by calcium and manganese (and, as we shall see in the case of a few vesicles, by zinc), and the extracellular fluids by calcium, copper, and molybdenum. The procedure is very like that of the analytical separation of elements by solvent extraction and/or precipitation followed by the transfer of separated phases. At each membrane selective pumping directs elements into selected compartments. The fold energy of the proteins which capture the metals are now seen to be used in several ways: (1) To generate thermodynamic selectivity. (2) To control exchange rates (see below). (3) To heighten catalytic significance, the entatic state. (4) To undergo conformational switches required, for example, in cation pumps-that is, to have two states of different binding strengths. (5) To permit recognition at membrane surfaces. The
selectivity
of metaliothionein
PI Metallothionein is not a usual protein. It is highly basic, has plenty of thiolate groups and no aromatic residues. It does not fold in the absence of metal ions. When bound to metal it folds. This means that the precise fold is not decided by the sequence but by the metal ions which it binds. The major metals associated with it in biological systems are zinc, cadmium, and copper. The fold with zinc is unlikely to be very similar to that when copper is bound, since copper (I) is much larger and copper (II) demands a very different stereochemistry. Receptor sites on membranes can bind the stereochemitally different metal-protein complexes selectively when it is possible that copper can be eliminated from the cytoplasm while zinc is retained to a large degree. This is the opposite use of a protein to that of transferrin, which very Aelectively introduces iron into cells and can also carry manganese. Metallothionein then has two functions, homeostasis of copper and zinc at very different cytoplasmic levels. (It also aids the removal or elimination of cadmium). Finally, in the folded form it binds to either RNA or DNA, through its basic amino acid side chains, leading to feed-back control over the production of the protein. The fact that metallothionine has a low fold-energy but a high binding constant, while the zinc enzymes have high fold energies and high binding
constants, is a consequence of their different protein sequences. Apart from binding energy differences these different fold energies control exchange rates, since on its own zinc exchanges relatively rapidly from inorganic complexes. The control by different proteins over exchange is undoubtedly a major feature of zinc biochemistry which we will examine below. Reversible
and irreversible
binding
All the known zinc enzymes can be isolated virtually without loss of zinc. This means that in the cell cytoplasm zinc enzymes are not able to exchange zinc freely and cannot be regulated by zinc exchange, although they can be specifically inhibited by zinc-binding reagents. Zinc exchange from metallothionine is relatively fast, however, so that zinc metallothionine can be a part of a regulation system but it is not yet clear what zinc functions it regulates. Clearly, it cannot directly affect the many irreversibly formed zinc enzymes which are produced in vesiclesfor example carboxypeptidase or the irreversibly formed cytoplasmic enzymes-unless there is rapid protein turn-over, which is not known. A clue to the real value of zinc regulationthat is of metallothionine-is to be found in the requirements for zinc in vesicles (Table l), where it is in very high concentration and can be exchanged readily. Zinc in vesicles
We have now seen how zinc enzymes can be preferentially formed in the cell cytoplasm but zinc proteins are also formed in special vesicles-for example, the islets of Langehans (insulin) Pollen
Sperm
Figure 4 Diagrammatic picture of the heavy concentration of zinc in gametes. ZnPl, an inhibitor enzyme, acrosin, in the vesicles of the sperm head; ZnP,, a keratin-like tail. Zinc in the tube of pollen may well be to protect RNA, while in the nuclear region it protects DNA.
and the vesicles of the pancreas which contain digestive zinc enzymes (peptidases, amylases, and lipases). The movement of protein through the Golgi membrane to the interior of these vesicles needs an unfolded protein which cannot carry zinc. It follows that some Golgi vesicles (which become the islets of Langehans or digestive vesicles) must specifically pump in zinc. This is possible, of course, once the copper has been sufficiently removed from the cytoplasm by metallothionine. Now the free zinc concentration in some of these well-known vesicles must be high (alO-*M) relative to that established by metallothionine in the cytoplasm (lo-‘*M) since the binding constants of zinc to insulin or cysteine, for example, at pH7 are low (Table 1). What do we know about such vesicles? For the most part, the answer is very little but knowledge is accumulating about the very special cases of the reproductive gametes and associated vesicle systems (figure 4) 17-121. There appear to be two different zinc fractions in both sperm and pollen [8, 121. One, the larger, is associated in sperm with the tail of the male gamete, which is made of keratin. As far as can be judged the function of the zinc here is to bind to sulphydryl groups partly cross-linking the keratin and helping to avoid oxidative damage to it. This is very similar to the funciton of zinc in hair and nails. This part of the zinc is really extracellular, since the sperm are held in a fluid space which is to be ejected and the tail is air extension of the male cell outside its cytoplasmic membrane. (There is some similarity with the insulin vesicles). The zinc in the head of the sperm cells is intracellular and some is probably in a special vesicular system said to be associated with m-RNA and/or a lysozomal vesicle. The situation is clearer in pollen [12]. The developed pollen cell has two zinc regions: a long tube through which the male DNA will pass and a head which retains the DNA. There is at the tip of the tube a high concentration of zinc in association with a vesicular recticulum which also carries messenger RNA. The idea that zinc actually binds this RNA is improbable, since it is known that all RNA has very poor zinc-binding capability. It is more likely that the m-RNA is associated with a protein which protects the RNA and prevents transcription. A reasonable guess would be that this is a thiol-containing protein to bind zinc and that the zinc protein folds so as to bind the m-RNA when the protein becomes similar to metallothionines which have a very positive surface (compare histones) and they do bind to DNA. Such proteins could protect RNA and DNA as is required 69
TABLE 5
SOME SYSTEMS
Process
Molecular
Nerve growth factor Keratin-like sperm tail* Sperm protease (acrosin) DNA & RNA condensations Spore germination Prohormone hydrolysis?
Zinc Zinc Zinc Zinc Zinc Zinc
*Keratin
filaments
BY ZINC
characteristics
prevents protects inhibits proteins release controls
111 El
enzyme activation from break-down activation stabilise very early enzymes?
171
P31 191 111
may also have a major role inside cells and even in cell division.
in condensed genetic material in gametes. There is also much zinc in association with the DNA in the head of the pollen. Returning to the mammalian reproductive system, it is known that during sperm ejaculation the sperm is surrounded by a fluid from the prostrate which is very rich in zinc and citrate compounds [ 10-111. The zinc/citrate solution seemingly adds further protection to the sperm, especially the tail, maintaining as long as possible the zinc and a reducing environment. This environment is (slowly) lost in the female reproductive tract, where the zinc is in relatively fast exchange and low concentration. The zinc may well be associated with citrate in vesicles before ejection. The zinc is in fast exchange, since zinc citrate is a relatively weak complex [ll]. Another example of zinc in fast exchange is the zinc cysteinate of the vesicles of the tapetum lucidem of the eye. Here zinc must be in fast exchange since zinc cysteinate is not so very stable at pH7. The free zinc is probably lo-‘M. Another parallel is to be found in the venom-containing vesicles of snakes which contain NGF, nerve growth factors (enzymes), in a protected state. In some cases it has been demonstrated that the zinc of NGF acts to stabilise the NGF precursor zymogen. Relatively easy loss of zinc liberates the NGF-enzyme but addition of zinc inhibits the conversion of the pro-enzyme. NGF, like the zinc cysteinate, is stored in a vesicle. These several observations on vesicle or specially protected cell systems (Table 5) lead to a new hypothesis for a major role of zinc. It is a protective/ trigger agent in pumped storage sys-
70
INHIBITED
tems in association with small chelates or proteins often containing thiols. It is a trigger in that it prevents reactions of DNA, RNA, or proteins (Table 5) until they are required. The zinc trigger is then the local drop of zinc concentration after release from the vesicle. By controlling enzymes, zinc can stabilise zymogens (nerve growth factor), and hormones (insulin) through stabilising prohormones and/or through polymerisation. Zinc pro-insulin is then a zinc buffer to prevent insulin activation in two ways. Zinc may also control the folds of RNA or DNA in inhibited forms in association with thiol-binding groups of amino acids in vesicles. The homeostasis of zinc in cell cytoplasm is then to be associated with the pumping of the zinc to high levels in many special vesicular systems. By contrast, the zinc in the normal cell cytoplasm is in a separate kinetic compartment where it is largely in irreversible complexes and required for fast catalysis of reactions of low specificity. Comparison with calcium is instructive. Free calcium is the biological trigger of fast reactions and its binding to proteins gives activation either in the cell or outside it. Free calcium ions are stored. In the case of zinc, bound zinc complexes are stored and activity is triggered by dilution which releases the reagents from zinc. If this is true we must search for zinc-containing vesicles in many cells. It may well have a special relationship with cell-growth processes, for example in reproduction and in brain [13], and together with calcium it may provide a very general set of control reactions. Finally, zinc may associate with keratin-like filaments both inside and outside cells.
References
[l] Sigel, H (ed.) ‘Zinc and Its Role in Biology and Nutrition’, Vol. 15 of ‘Metal Ions in Biological Systems’, Dekker, New York, 1983. [2] Williams, R. J. P. FEBS Letters, 703, 87, 1982. [3] Vallee, B. L. and Falchuk, K. Phil Trans. Roy. Sot. London, 294B, 185, 1981.
[4] Kagi, J. H. T. and Nordberg, M. ‘Metallothionine’, Birkhauser Verlag, Base], 1981. [5] Dennard, A. E. and Williams, R. J. P., J. Chem. Sot., (London), 1966A, 612 [6] Vallee, B. L. and Williams, R. J. P. Proc. Natl. Acad.
Sci. (U.S.),
59,
498, 1968. [7] Steven, F. S., Griffin, M. M. and Chantler, E. N. Int. J. Andrology, 5, 401, 1982.
[8] Roomans, G. M., Lundevall, E., Bjorndahl, L., and Krist, U. Znternut. J. Andrology, 5, 478, 1982. [9] Johnston, K., Stewart, G. S. A. B., Scott, I. R., and Ellar, D. J. Biochem. J., 208, 407, 1982.
[lo] Mann, T. and Lutwak-Mann, C., ‘Male Reproductive Function and Semen’, SpringerVerlag, Berlin 1981. [ll] Arver, S., Acta Physiol. Stand. 116, 67, 1982. [12] Ender, C. H., Li, M. Q., Martin B., Park B., Nobilung R., ReissH-D., and Traxel K. Protoplasm 1983, submitted. [13] Itoh, M., Ebadi, M., and Swanson,S., J. Neurochemistry, 41, 823, 1983.