Zinc Enzymes I. Bettini', C. Luchinat, and R. Monnannl University of Florence. Florence, Italy Zinc is an essential element-one that is necessary for the occurrence of reactions that are required in the metabolic processes of living organisms. I t is the second most abundant transition or post-transition metal. I t is transported by proteins (macroglobulin, transferrin, and albumin), stored in a protein (thionein), and bound to proteins. I t is generally bound to histidines, carboxylate-containing residues, and cysteines. In a crude way, zinc can be classified according to its degree of direct involvement in the catalytic mechanism. If so, it may coordinate a substrate molecule and activate it for the required reaction. Independently of this classification zinc acts as a Lewis acid; i.e., it accepts lone pairs by donor groups; its Lewis acid properties are primarily important when it acts as catalyst.
Figure 1. Stability constants of trisalhylenediaminometal (11) and tenabromp metallate (11) complexes.
Zinc Is Bound to Proteln Residues The first question that arises is why nature has chosen zinc for this Lewis acid role. Among hipositive 3d metal ions plus zinc, a relative scale of Lewis acidity can he obtained by comparing the formation constants of their complexes with a given ligand, e.g., 1,2-diaminoethane ( I ) (en) (Fig. 1). The actual reaction is, however, a substitution reaction of the coordinated water rather than a direct measurement of the Lewis acidity through the reaction
+
M2+ 3 en'
M(enh2+
(2)
With this limitation in mind, it appears that copper(I1) and nickel(I1) are better Lewis acids than zinc(I1); however, if the reaction is referred to the formation of tetrahedral compounds, e.g. then zinc(I1) is relatively a better acid (2).Indeed in nature, zinc(I1) is often four or five coordinated; furthermore, zinc forms four., five-, and six-coordinated complexes with comparable stability, and ligands are kinetically labile. Both are important features when a metal ion behaves as a catalyst. Finally, zinc is stable only in the coordination number +2 and, therefore, cannot perform redox reactions. All of these reasons make zinc(1I) the best catalyst when the role of the metal ion is that of being a Lewis acid. Nomatalytlo Zlnc(ll) When zinc-containing proteins do not have any enzymatic role, it is easy to classify the ion as a structural ion in the sense that it stabilizes the entire structure of the protein. In enzymes, zinc can be safely classified as structural when it stabilizes the quaternary structure, i.e., it keeps together subunits of a protein. A typical example is provided by aspartate transcarbamylase, which consists of 12 polypeptide chains (3).Zinc is hound to four cysteinyl residues in each of six of the 12 polypeptides (Fig. 2A). Its presence is
'Mailing address: Department of Chemistry, University of Florence. Via G. Capponi 7, 50121 Florence, Italy. 924
Journal of Chemical Education
Figure 2. Noncatalytic zinc In: (A) aspartate transcarbamylase. (8)horse liver alcohol dahydrogenase,arm (C)bovine erythrocyte superoxide dismutase.
required for stabilization of the quaternary structure (4). In other cases, the classification of zinc(I1) as noncatalytic in enzymes often follows our incapability to assign the ion a specific role. For example, liver alcohol dehydrogenase (LADH) contains a zinc ion that is called catalytic and another one, 20 A away, that is noncatalytic because the enzyme in crvstalline form works without it (5).Analogous to aspartate ~onscarl,amylase, the latter is coordinated b y four cysreineresiducs (Fig.?B). What is it3n)le? I'ossihly it is not unique, but certainliit renders the enzyme more stable and more functional. In alkaline phosphatase (AP) there is a t least another metal site, besides the catalytic site, which is occupied by zinc. A role of this metal ion is that of increasing the catalytic efficiency, and i t may also stabilize the tertiary structure (6). Again in superoxide dismutase (SOD) zinc d. a v.s a structural role. as lone as the irreeular coordination around copper(l11 has a biological meaning. Such irregular srereochemistrv is reached with the helo of zinr. which is bridged to t h e copper(I1) ion through histidinato residue (Fig. 2C) (7).Zinc increases the thermal stability of SOD (8). The discovery of the fine properties induced by noncatalytic zinc is a perspective in the study of zinc enzymes. Catalytic Zlnc ZinrrIl) is present in the ratalytir siteof many enzymes; if zinc is removed, the catalytir efficienry drops to zem. Zinc-
containing enzymes belong to all six classes of enzymes: hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferasea Most often, the role of zinc is to hind the suhstrate and activate it. For our purposes, the term suhstrate includes the solvent water in hydration reactions. For example, in carbonic anhydrase the water coordinated to zinc, which is further bound to three neutral histidine nitrogens (9),
has such a low pK,(IO) that at physiological pH the enzyme is largely in its active form (11)
Carbon dioxide, which is proposed to he bound about 6-8 A from the metal ion (12), is then attacked by the coordinated hydroxo group to form a bicarbonate derivative
the attack to give an anhydride which is eventually hydrolyzed;
The possibility has been considered that a water molecule is still coordinated t o the metal together with the suhstrate and that in a transition state it becomes an hydroxo group which performs the nucleophilic attack. The activity of the enzyme depends on the pK, of the free Glu, which is around 5; when Glu is protonated, the activity drops to zero. The activity vanishes also a t high pH values with a pK. of 9. There is no agreement on the nature of a group with such pK., hut the coordinated water is a reasonable candidate. Indeed the coordinated hydroxide would not allow hinding of the substrate. In LADH the catalytic zinc ion is hound to a histidine and to two cysteines and is exposed to solvent (17). The enzyme requires the presence of NAD+ or NADH to assist with hydride transfer:
Even though the enzvme oxidizes alcohols to aldehvdes. the reverse reaction, e.g.. the hydrogenation oiaceialdehvde in nresonce of N h U H ii more illustrative. The liuhstrate hinds to zinc in a fashion similar to the hinding of peptides to CPA (18): This derivative is in equilibrium between four- and fivecoordinated species, the latter containing a water molecule, as shown through spectroscopic studies on the cohalt(I1) analog (13). At this point HC08- can he released followed by a proton in such a way that the enzyme is restored (14). The role of zinc is, therefore, that of providing a coordinated hydroxo group a t physiological pH; such a group is a better nucleophile than free water. Among the many zinc enzymes (200 from different species) carhoxypeptidase A (CPA) and LADH will also he discussed as representative examples. CPA cleaves the Cterminal peptidic bond, especially if the terminal amino acid contains an aromatic group. The enzyme contains in the cavity an arginine which hinds the terminal COO-, an hydrophobic moiety which binds the aromatic group of the terminal residue, and possibly other groups which help in orienting the suhstrate. Zincis bound toaglutamatereeidue, to two histidines, and to solvent (15). The suhstrate hinds the metal through the carhonyl bond, thus making easy a nucleophilic attack on the carbonyl carbon (16).
The carhonyl carbon is activated for a nucleophilic attack, which in this case is performed by H- provided by the coenzyme NADH which is properly oriented for this purpose. The resulting alcoholate
accepts a proton by an incoming water molecule and leaves as alcohol being substituted by an hydroxo group.
When NADf dissociates, the coordinated hydroxide takes up a proton. For the principle of microscopic reversihility, the steps in reverse order occur for alcohol dehydrogenation. In this case the enzyme is in its hydroxo form, the coordinated water having a pK, of 7.6 when the coenzyme NAD+ is present (19). The Effects ol Other Acidic Groups In the Cavity
Such an attack is possibly accomplished by a water molecule activated through hydrogen bonds with a Glu residue hanging into the cavity; or the Glu itself could accomplish
Proteins contain many acidic groups with pK.'s varying in the range 3-11. Therefore, any investigation of a pH-dependent property should consider that the protein is different a t Volume 62
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925
every pH since every solvent-accessible acidic group has a different protonation extent. However, the most dramatic effect on enzymatic activity is shown by acidic groups close to, and interacting with, the metal ion. For example, CA contains a histidine hanging into the active cavity; this residue has a pK. of 6.5 when free (20), hut i t interacts via hydrogen bonds with the Zn-OHnmoiety (21). The pK, of a water molecule coordinated to zinc in a dipositive tetrahedral complex can he estimated to he around 8, in the low limit of the observed values in model complexes. When the two acidic groups interact, we can figure out that a t pH 5 both are protonated
the anions NCO- and N3- for the diprotonated and monoprotonated species, as calculated from the electronic spectra of cobalt-substituted derivatives, is reported in the table together with the affinity values of CI-, which is necessary to solubilize the protein. I t appeam that the pH dependence of anion affinity in CPA is parallel (and similar in magnitude) to t. in ..t.h ..n -. ..CA. - - ..
In LADH, there is a strona anion binding site formed by Arg 4: and Arg 369 (2.5). ~ n h pret'erentiallg s hind at th&
sire rather than ar the zinc ion. Only a second anion ran bind the metal ion, though with an affinity one order of magnitude smaller (26). The lnvestlgatlon through Metal Substitution
Depending on the extent of the interaction, the coordinated water molecule lowers its pK, of several units due to the presence of a positive, protonated group. In CA the pK, of water in the presence of histidinium is around 6 (22). The pK, of histidinium, on the contrary, is little affected by the presence of water; however, i t is affected by the presence of the hydroxo group. In other words we can appreciate the pK, values of the-following reaction scheme
Most of the above information has been obtained on metal-substituted derivatives, since the natural zinc enzymes can he investigated with a quite small number of investigation devices. Zinc(I1) can he removed by treating the metalloprotein with a strong ligand like 2,6-dipicolinic acid or I,l0-phenantroline. Then other dipositive ions can be added which occupy the metal site. The cohalt(I1)-substituted derivatives are particularly. good models of the natural enzymes due to the similarity in the chemistry of the two ions. The activity of these derivatives is similar to that of the natural products. Cohalt(I1) has well defined electronic
Affinity Constants of Anions for CoCPA pK., = 5.3 K(Nc), = 1670 K(NC0-)I = 1640 K(CIT), = 9.6
p&. = 8.9 K(N,-), = 1 14 K(NC0-)I = 6 1 K(C1-), = 0
With these pK.'s, we can calculate that the share of the Zn-OH species is 40% a t pH 6; this value accounts for the pH dependence of the activity of the enzyme (23). Anions are inhibitors of t h e enzyme, since they give rise to derivatives of the type
which prevent hicarbonate from hinding. The affinity of Lewis bases like anions for a hiacidic group dramatically depends on pH. If we assume that there is only one hinding site, i.e., the metal ion, we can determine an affinity constant for the hiacidic species (K = 10,200for Nos-) and another, quite lower, for the monoacidic species (K = 250) (22). The effect of a charge in the cavity is possihly quite complex, hut one can expect an electrostatic effect on a charged inhibitor, In CPA, t h e affinity of NCO- and Ns- is governed by the two pK.'s involved in the activity (24). The affinity of 926
Figure 3. Electronic spectra at various pH (increasing with Increasing of the absorption at 840 nm) of Co(l1) substituted erythrocyte carbonic anhydrases: (A) Wvine isaenzyme 11. (0)human ixlenzyme 11, (C) human isaenzyme I, and (D) bovine isoenzyme Ill.
Journal of Chemical Education
Il3Cd has also been widely used and monitored through NMR. Although this metal ion is not an ideal substitute for zinc, the observed chemical shifts can be related to the nature and number of the donor atoms (Fig. 4). Perspectives
Even though much is known on zinc enzymes, a great deal of data of the kind discussed above is still necessary to understand significant details of the catalytic mechanisms. Evenmore information can he eained with the helnof molecular biology. Enzymes without certain amino acids in strateeic oositions can be ~ r e ~ a r eand d . their chemical and catarytie properties should be in"estigated and compared with those of the natural enzymes. This era has already begun.
-
Llierature Cited (11 Bssalo. F.. and P e s w n . R. 0.. "Mechanism. of InarganicReactions: 2nd ed.. Wiley and Sons. NY. 1967. I21 Bianchi, A,, and Pauletti, P., Inorg. Chim. Acto, 137.96 (19851. (31 Monaco.H. L., Crawford, J . L.. and Lipscomb, W. N., P?oc.Noll. Aeod. Sei. USA. 75. 315 (1972). (51 Sitkouski, A., and Vallee, B. L., Biochamialry, 17,2850 (19781, (61 Coloman,J. E.,andGettins.P.,Adu. Enrymol.,55,381 (198XI. 171 Tainer.J. A . Getzoff, E. D.,Beem, K. M.,Riehardson, J. S.,sndRichardson, D. C., J. a"", ,.,,., n;"< ,C" 7n, ..,.,, .,., ,,Sam ,, ,, bt Furmnn H I .and tr.d.\,.h.l . J h I r h . n 2lr.2611 19-71 .*I IG,,nnn K t i , n ' H,,lyn,.lr* "Ud Phvrlol.,g\ 8 ,',,Lon U4. Xldt." IF"',^‘ Baucr.
,.".",.
Figure 4."'Cd NMR s p e c t r a of: Cd, liver alcohol dehydrogenase (l), a n d in p r e s e n c e of imidazole (2).a n d NADH (3)(37);Cd. superoxide dismutase (4). a n d CdrCul superoxide dimutase (5)(38):cadmium carbonic anhydrase as a function of pH (61, human I, human 11, a n d bovine I1 isoenzymes, a n d adducts with cyanide a n d bicarbonate (7) (39): cadmium carboxypeptidase A, P phenylpropionate adduct (8)(40); Cd, alkaline phosphatase, catalytic s i t e s (9). Cd2 monophosphoryl derivative (10).CdrMg2 monophosphoryl derivative or Cd2Mg2 diphosphoryl derivative (11). a n d Cds diphosphoryl derivative (12) (41).A. 8 , and C indicate t h e t h r e e metal binding sites.
spectra which allow researchers to monitor the pH-dependent properties and interactions with substrates and inhihitors. ~ h electronic k spectra of various cobalt-substituted carbonic anhydrase isoenzymes are shown in Figure 3. Another characteristic of this kind of cobalt(I1) is that the NMR signals of protons of residues coordinated to the metal ion can he safely detected outside the diamagnetic protein region. When the spectra are recorded in D20, the signals of the exchangeable protons disappear; among these there are the histidine NH's. In this way the coordinated histidines can be counted and monitored easily under the various chemical conditions (27-30). Manganese(I1) and copper(I1) derivatives are also often studied (311, although the latter does not show any activity and the former onlv slight activitv. Nevertheless. thev Drovide structural inf&ation, mainly through EPR ($2,'33) and NMR (34-36) relaxation studies.
I131 Bertki, I., Lanini, G., and Luehinal, C., J. Amw. Cham. Sor., 105,5116 (19831. (141 Venkatasuhban, K. S., and Silverman, D. N., Biochemistry, 19.1984 (1980l. (151 Reea. D. C ,Lewis, M.. Honratko,R. 6.. Lipscomb, W. N , s n d Hardman. K. D,Pmc. Noti. A c d Sci. USA, 78.3408 (19811. (161 Vsllee. B. L.. Galdes, A,, Add, 0. S.. and Riordan. J. F., in "Metal lona in Biology: (Editor: Spiro, T. G I , Wileysnd Sons. NY. 5.25 (1983). I171 Eklund. H.. and Branden. C. I., in "Motel Ions in Biology: (Editor Spim, T. G.), Wiley and Sons. NY. 5,123 (1983). I181 Dunn,H.F.,Dietrich,H.,MscGibbon,A.K.,andZeppezsuer,M..J.Inarg.Bioch~m., 14.297 l19RLIDietrieh,H.,Maret, W.,and Zeppemuer,M.,Eur.J. Biochem., 100, 267 (19791. (19) Kvamman, J., and P e t t e r n , G., Eur. J. Bioehem., 100, 115 (1979). Andernson, P., Kvsraman,J.. Lindstrbm, A..olden,B.,andPeberason,G.,Eur. J. Riochem., 113, 125 (19811. I201 Campbell, I. 0.. Lindskog, S., and White. H. I., Biochim. Biophyr. Acto, 484, 443 (19771. I211 Lindskng. S.. lbrshim. S. A , Jomson, B. J.. and Simonsson, I., in "Tho Cwrdination Chemistry of Metslloenzymes..' (Editors: Bertini, I., Orago. R. S., and Luahinat. C.!, Reidel. D.. Donlrecht, Holland. 1983, p. 49. (22) Bertini, I.. Dei, A,, Luchinat, C., and Monnanni, R., Inor8 Chem., 24,301 (19851. I231 Simons~on,I., and Lindrkog, S.. Eur. J. Bioehem., 29,123 (19811. 1241 Bmtini, I.. Lsnini. G.. Luchinat, C.,and Monnanni, R., l n o l g Chim. Acto, 107, 153 (19851. I261 Andersson, I.. Zepperauer,M.. Bull.T.,Einar~son,R.,Norne,J.-E., andLindman,B., Biochemistry, 18,3407 (19791. (26) Bertini. I., Zepperauer, M. et sl., in preparation. 127) Bertini, L. Canti, G.. Luehinat, C , end Meni, F.. J. Amer Chem Soc, 103, 7784 (1981l. I281 Beriini, I., Canti, G., and Luchinat, C., J. Amar. Chem. Soc.. 104,4943 11982). W..Rawcr,J.,endZeppezsuer, I291 Bertini,I..Garber,M..Lsnini,G.,Luehinaf,C.,Maret. M., J.Amer. Cham,Sac., 106, I226 (1984). (30) Bertini. I.. and Luchinat, C., Ad". Inorg. Bioch~m.,6.71 (1965). 1311 Bertini. I., and Luchinat, C., in"Meta1 Ions in Biological Systems: (Editor Sigel. H.I.Dakker Lnc.. NY IS. 101 119841. 132) Rosembeq.R C.,RooL C.A..Ber~st(ii,P.K.,andGrw,H.B., J.Ame,. Chem. Sw., 97,2092 (19761. (33) Shulman. R. G., Navon, G.. Wiluda, B. J.. Dauglss~.D. C.. end Yamme, T.. Proc. Noti. Amd. Sci. USA. 56.39 (1966l. I341 Kushnir,T..and Nav0n.G.. J.Mn#.Reson.,56.373 (L984l. (351 Ko~nig,S.H.,and Brown,R.D..Ann.N.Y.Acmd.Sci. USA,222,752 (~973I.Koenig. S. H.. and Blown. R. O., in"ESR and NMR of Paramagnetic Specie8 in Bialogical and RalatedSystams,"lEditors:Bertini,l.,andDrago,R.S.l,RoidelD., 1980,p.89. I361 Bertini, I., Briganti. F., Luchinat, C., Maneini. M.. and Spina. G., J. Mag. Res, 62 119%)~
(571 Bobsein, B.R.,andMyers,R. J., J.Biol. Chem.,256.5313 (1981). (36) Bailey, D. B., Ellis. P. D.. and Fee. J. A.,Bioehamiairy, 19.591 (19801. (39) Johnrson, N. B-H.. Tibell. L. A. E.,Evelhoch, J. L.,Rell, S. J..andSudmeier, J.L., Pruc. Noti. Acod. Sci. USA. 77,3269 (19WI. (40) Armitage, I. M., Seboot Uiwkamp. J. M., Chlebowski. J. F., and Coleman, J. E., J. Map". &*on. (41) Otvos, J. D., an
