The Role of Metals in Enzyme Activity*

Home , Lability, Trace metal, Xanthine oxidase

JAMES F. RIORDAN, Ph.D.

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and Division of Medical Biology, Veter Bent Brigham Hospital, Boston, MA 02115

ABSTRACT

Metal ions play important roles in the biological function of many en zymes. The various modes of metal-protein interaction include metal-, ligand-, and enzyme-bridge complexes. Metals can serve as electron donors or acceptors, Lewis acids or structural regulators. Those that participate directly in the catalytic mechanism usually exhibit anomalous physicochemical characteristics reflecting their entatic state. Carboxypep- tidase A, liver alcohol dehydrogenase, aspartate transcarbamoylase and al kaline phosphatase exemplify the different roles of metals in metalloenzymes while the nucleotide polymerases point to the essential role of zinc in maintaining normal growth and development.

Introduction At present, reliable measurements of small concentrations of metals present in Certain metals have long been recog tissues, cells, subcellular particles, body nized to have important biological func fluids and biomacromolecules can be tions primarily as a consequence of nu performed by colorimetry, fluorimetry, tritional investigations.14,15,22 Thus, the polarography, emission spectrometry absence of a specific, essential metal with spark, flame or plasma excitation from the diet of an organism invariably sources, x-ray and atomic fluorescence, leads to a deficiency state characterized atomic absorption and neutron activation by metabolic abnormalities with altered analysis, among other methods. Metals or retarded growth. Because such metals that have been detected by such are usually present in tissues in very techniques and currently known to be small amounts it was reasonable to sus components of metalloenzymes include pect that they might play a catalytic role, cobalt, copper, iron, manganese, molyb perhaps participating in enzymatic reac denum, nickel, selenium and zinc (table tions. The actual discovery of metalloenzymes, however, required the availability I). Aside from its role in vitamin B 12, of accurate, sensitive, analytical method cobalt, to date, has been found to be a ology. As a consequence, the unequivo cal demonstration of a role for metals in * Supported by Grant-in-Aid GM-15003 from the enzyme action is of relatively recent vin National Institutes of Health of the Department of tage. Health, Education and Welfare. 1 19 120 RIORDAN

TABLE I as well as a molybdoferrodoxin, a com Metals Present in Naturally Occurring Metalloenzymes ponent of the nitrogenase system of nitrogen-fixing bacteria Azotobacter vin- Metal Enzyme Function elandii and Clostridium pasteurianum-15 Nickel has been found to be present in Cobalt Transcarboxylation Copper Oxidoreduction urease 50 years after the enzyme was first Iron Oxidoreduction crystallized.8 Selenium, which has been Manganese Various Molybdenum Oxidoreduction recognized as an essential nutrient for Nickel Urease Selenium Peroxidase more than a dozen years, has recently Zinc Various been shown to be a component of an en zyme, glutathione peroxidase from eryth rocytes, the first example of a selenoen- component of but one enzyme, the zyme.10 biotin-dependent, zinc-containing Zinc enzymes are among the most oxaloacetate transcarboxylase of Pro common of the metalloenzymes number pionibacterium shermanii.19 Copper is ing over 70 and representing each of the present in a large number of enzymes six categories of enzymes designated by that catalyze oxidoreduction reactions the International Union on Biochemistry such as tyrosinase, lysyl oxidase and (IUB) commission on enzymes (table II). cytochrome oxidase.20 Iron is also found primarily in enzymes that participate in Zinc metalloenzymes exhibit perhaps the oxidoreduction reactions; in addition, it greatest diversity both of catalytic func tion and of the role played by the metal plays a major role in oxygen transport.18 Manganese has been identified as a com atom.15,21,22’23,27'28,30 The metal is present ponent of pyruvate carboxylase from in several dehydrogenases, aldolases, chicken liver and is present in Es peptidases and phosphatases. Zinc en zymes participate in carbohydrate, lipid, cherichia coli superoxide dismutase.15 It also serves as an activator for many protein and nucleic acid synthesis or de metal-activated enzymes; however, in gradation. Several examples of zinc en most of these cases, magnesium and other zymes will be cited to illustrate the role divalent cations can fulfill the same func metals in metalloenzymes and the gen tion. eral importance of zinc to metabolism. Other metals such as sodium, potas Molybdenum is found most frequently sium, calcium and magnesium can also in flavin-dependent enzymes, usually in conjunction with non-heme iron and assist in the action of enzymes. With th acid-labile sulfur. A typical example is ese, the mode of metal-enzyme interac xanthine oxidase. A molybdoheme pro tion is complex and often difficult to es tein, sulfite oxidase, has been described tablish. Still other metals, such as chromium, vanadium and tin, have been TA BLE II shown to be either essential for growth in Currently Known Zinc Metalloenzymes certain species or components of biologi cal macromolecules. However, their rela International Union tionship to enzyme mechanisms has not o f Biochem istry System Number Example been established.

E.C.l Oxidoreductases 7 Alcohol dehydrogenases Enzymes affected by metal ions have E.C.2 Transferases 8 DNA polymerase been operationally defined as either E.C.3 Hydrolases 23 Carboxypeptidase E.C.4 Lyases 19 6-ALA dehydratase metalloenzymes or metal-enzyme com E.C.5 Isomerases 1 Mannose-P isomerase E.C.6 Ligases 1 Pyruvate carboxylase plexes.28 A metalloenzyme contains a firmly bound, stoichiometric quantity of a ROLE OF METALS IN ENZYME ACTIVITY 121 metal as an integral part of the protein with the role of metals in metalloen molecule. Removal of the metal by treat zymes and will not attempt to cover the ment with chelating agents, for example, interesting but voluminous literature de abolishes catalytic activity. In instances aling with metal-enzyme complexes. where the resultant apoenzyme is struc- tually stable, restoration of the metal can The Interaction of Metal Ions regenerate full biological function. In With Enzymes contrast, metal-enzyme complexes are A number of schemes have been pres more loosely associated, the criterion for association being metal activation of ented24 to describe the types of interac tions that can occur between metals, en catalysis. The metal ion is frequently not zyme proteins and substrate (or inhibitor) an integral part of the molecule when iso (figure 1). The first of these represents an lated, and the enzyme may exhibit partial interaction between the substrate and the activity in the absence of the metal ion. metal ion to form a complex that acts as Obviously, the difference between these two classes of metal-enzyme systems de the true substrate. Substrate-metal com- pends on the magnitude of the metal- plexation can occur prior or subsequent protein stability constant which can be a to the formation of the enzyme-substrate complex. This type of behavior is typi function of the metal atom as well as en cally observed with metal-activated en vironmental conditions such as pH, buf zymes. The second scheme indicates that fer and ionic strength. The metalloen- the metal first binds to the protein and zymes are better suited for elucidation of then serves as a site of interaction with the metal protein interaction and for ex trapolating such information to the un substrate. In this instance, the metal can function either as a binding site, as a derstanding of enzymic mechanisms. component of the catalytic apparatus of Moreover, they lend themselves more the enzyme or both. readily to a definite assessment of the An example of both such possibilities is physiological role of the metal. Metal- given by the role of zinc in carboxypep- enzyme complexes, however, have been tidase A. Here the zinc atom is believed of great theoretical importance in the un to interact with a peptide substrate via derstanding of catalytic phenomena and the carbonyl oxygen atom of its terminal general mechanisms of catalysis by metalloenzymes. M + S = MS At present some 2,000 or more different enzymes have been isolated and charac E + MS = EMS terized and it has been estimated that at least one-third of these require or contain metal ions.14 In fact, the actual number of metal-dependent enzymes may be even 2) E + M = EM greater for it has been pointed out that } “There probably does not exist a single EM + S = EMS enzyme-catalyzed reaction in which either enzyme, substrate, product, or a combination of these is not influenced in 3, E + M = ME a very direct and highly specific manner by the precise nature of the inorganic } ME + S = MES ions which surround and modify it” .17 F i g u r e 1. Interactions between metal (M), en This paper will be concerned primarily zyme (E) and substrate (S). 122 RIORDAN peptide bond, i.e., the one that is suscep iron-promoted decomposition of hydro tible to hydrolysis. However, even gen peroxide although in this case though some kind of metal-substrate catalase is at least a million times more bond may be formed, the metal does not effective than iron alone. Thus, the pro appear to be essential for peptide sub tein component of a metalloenzyme con strate binding. Peptides bind to the tributes many of the critical aspects of the metal-free apoenzyme as well as they do catalytic mechanism. to the metalloenzyme, even though they Zinc, on the other hand, does not are not hydrolyzed.2 Thus, for peptide undergo a change in oxidation state dur substrates the metal presumably serves ing enzymatic catalysis even though it as a catalytic site. On the other hand, participates in oxidoreduction reactions, ester substrates of carboxypeptidase do e.g., as a component of alcohol dehydro not bind to the apoenzyme. It has been genase. The zinc cation has a stable, d10 proposed that differences in the mode of electronic configuration and has little interaction between substrate and metal tendency to accept or to donate single account for the numerous kinetic differ electrons. Instead, it serves as a Lewis ences that have been observed for car acid interacting with electronegative boxypeptidase acting on ester and pep donors to increase the polarity of chemi tide substrates, respectively.2 cal bonds and thus promote the transfer A third scheme would have the metal of atoms or groups. Substitution reactions acting at a site on the enzyme remote of simple metal chelates generally pro from the active site. In such instances, ceed via intermediates with an open the metal could either serve to maintain coordination position or a distorted coor protein structure and only influence dination sphere. Zinc (and also cobalt) catalytic activity indirectly or else it can readily accept a distorted geometry could regulate activity by stabilizing and, hence, would appear to be well more or less active conformations of the suited to participate in substitution reac protein. The latter situation would more tions as, for instance, in carbonic anhyd- likely pertain for metal-activated en rase, carboxypeptidase and alkaline zymes where the metal-protein interac phosphatase. tion is more readily controlled by manip ulation of the ambient metal ion concen Entasis and Metalloenzyme Active Sites tration. It should be emphasized that What then is the role of the protein in these schemes are not all mutually exclu the mechanism of action of a metalloen sive and that some metalloenzymes are zyme? As indicated for catalase, it makes known to contain functionally different a major contribution to the enhancement classes of metal ions. of reaction rate. It creates a proper bind ing locus to ensure substrate specificity. The Role of Metals in the Mechanism It juxtaposes catalytic residues in the of Catalysis precise orientation with respect to the Iron, copper and molybdenum are most susceptible reaction centers of the sub commonly encountered in enzymes strate. It provides a suitably balanced catalyzing oxidoreduction reactions. In hydrophobic-hydrophilic environment the majority of cases, the metal ion par and serves to collect all the participating ticipates directly in the electron transfer species in reactions between several process and undergoes a cyclic change in molecules. It also provides liganding oxidation state. Oftentimes the free metal groups for binding the metal. The is capable of catalysis by itself as with the number, nature, orientation and im ROLE OF METALS IN ENZYME ACTIVITY 1 2 3

mediate chemical environmental of these groups will dictate, in large part, the chemical characteristics of the bound metal ion. It is this total combination that manifests as the catalytic activity of the enzyme. In other words, the protein con tributes a constellation of ligands at the metal binding site that prepares the metal for its catalytic role. Prior to the interaction with substrate the protein has already poised the metal for catalysis. In the case of iron or copper, for example, the metal may be held in a compromised geometry between those normally assumed by its two oxidation FIGURE 2. Visible absorption spectra of E. coli states and approximating that of the alkaline phosphatase containing 4 g atoms of cobalt plausible transition state for the reaction per mole of protein compared with spectra of cobalt in which it is involved. Vallee and Wil model complexes. liams have called this an entatic state im plying a state of tension at the active site physicochemical properties are difficult of the enzyme.32,33 It has been defined by to assess. However, in virtually every them as “the existence in the enzyme of zinc metalloenzyme where it has been at an area with energy, closer to that of a tempted, the zinc ion can be replaced by unimolecular transition state than to that cobalt to form an enzymically active de of a conventional stable molecule, rivative with a visible absorption spec thereby constituting an energetically trum.29 The resulting spectra differ signif poised domain.” icantly from the spectra of model cobalt (II) complexes as shown for alkaline Spectral Properties of Metalloenzymes phosphatase in figure 2. Moreover, altera tion of the coordination sphere by the ad On the basis of entasis, the dition of another ligand such as an in- physicochemical properties of metals in

metalloenzymes might be expected to di T A BLE III ffer from those observed for the same metals present in well-defined model Activities of Metallocarboxy-Peptidases coordination complexes. Both the absorp Activity (v/vzinc X 100) tion and EPR* spectra of copper and Peptidase* Metal Esteraset nonheme iron enzymes are unusual (v'vzinc x 100> when compared to those of simple copper Apo 0 0 and iron complexes.32,32 In particular the Zinc 100 100 so-called copper blue enzymes exhibit Cobalt 200 114 Nickel 47 43 absorption bands that differ strikingly in Manganese 27 156 intensity and fine structure from those of Cadmium 0 143 Mercury 0 86 non-catalytic copper proteins. Rhodium 0 71 Lead 0 57 The zinc ion has neither intrinsic color Copper 0 0 nor unpaired electrons hence its

*0.02 M benzyloxycarbonylglycyl-L-phenylalanine, pH 7.5, 0° C. tO.01 M benzoylglycyl-DL-phenyllactate, pH 7.5, * Electron spin resonance. 25° C. 124 riordan

The cobalt enzyme, for example, has twice the peptidase activity of the zinc enzyme while the nickel and manganese enzymes are much less active. The pep tidase activity of cadmium carboxypep tidase is a function of the particular pep tide substrate examined. In most cases, it is usually less than a few percent of that of the native zinc enzyme. Mercury, rhodium, lead and copper carboxypep- tidases are essentially inactive as pep tidase. A comparison of the kinetic parameters for the zinc, cobalt, man ganese and cadmium enzyme-catalyzed hydrolysis of benzoyl-glycyl-glycyl-L- phenylalanine (table IV) reveal a range of kcat values from 6000 min”1 for the cobalt enzyme to 43 min-1 for the cadmium en zyme.2 The Km values, on the other hand,

F i g u r e 3. Schematic representation of the are almost totally independent of the par mechanism of peptide hydrolysis catalyzed by car- ticular metal present. Thus, it would ap boxypeptidase A. pear that the primary role of the metal is to function in the catalytic process and hibitor anion converts the irregular spec that it has little to do with substrate bind trum to one closely resembling a regular ing. This is consistent with previous tetrahedral cobalt ion. It is important to studies showing that peptide substrates note that these properties can be ob bind to apocarboxypeptidase and prevent served in the absence of substrate. While the reassociation of the metal-free protein it has not been possible to interpret these with zinc. unusual properties of metals in metalloenzymes in terms of precise geometries X-ray analysis of carboxypeptidase and, ultimately, mechanisms of enzyme crystals together with amino acid se action, nevertheless they are quite con quence information has identified three sistent with views on the entatic nature of protein ligands to the zinc.416 They are active sites. glutamic acid-72, histidine-69 and histidine-196. Both histidyl residues are The Role of Zinc in Metalloenzymes held in position by hydrogen bonding to carboxyl side chains. In the crystalline T h e R o l e o f Z i n c i n state, a fourth coordination site is oc C arboxypeptidase cupied by water. Diffusion of the very Carboxypeptidase A is a classic zinc slowly hydrolyzed dipeptide, glycyl-L- metalloenzyme.31 It contains one g atom tyrosine, into the crystals gives an of zinc per molecular weight (34,500). enzyme-dipeptide complex. The car Removal of the metal atom either by bonyl oxygen atom of the substrate’s pep dialysis at low pH or by treatment with tide bond is thought to displace the coor chelating agents gives a totally inactive dinated water atom and interact directly apoenzyme.22 23 Activity can be restored with the zinc. This polarizes the carbonyl by readdition of zinc or one of a number bond and promotes an attack by the car of other divalent metal ions (table III). bonyl group of glutamic acid-270 at the ROLE OF METALS IN ENZYME ACTIVITY 12 5 carbonyl carbon atom either directly or T A B L E IV through a water molecule (figure 3). A Metallocarboxypeptidase - Catalyzed Hydrolysis key feature of the catalytic mechanism of Benzy1-glycylglycyl-L-phenylalanine porposed by the x-ray crystallographers is k - 1 Metal a 12A movement of tyrosine-248, a resi c a t (mm ) Km (mM 1) due thought to serve as a proton donor to Cobalt 6,000 1.5 the susceptible peptide nitrogen atom.16 Zinc 1,200 1.0 Manganese 230 2.8 Several conclusions drawn from Cadmium 41 1.3 studies carried out with carboxypeptidase in solution differ significantly from those derived from x-ray analysis. Using a the catalytic properties of both glutamic chemically modified derivative of car- acid-270 and tyrosine-248. The most im boxypeptidase in which tyrosine-248 was portant thing, however, is that zinc ion is coupled with diazotized arsanilic acid, central to the overall process of hyd Johansen and Vallee demonstrated that rolysis. Its major function is to be able to the azotyrosyl residue interacts directly undergo ligand exchange when triggered with the zinc ion thus constituting a by the entrance of substrate into the ac fourth and a fifth protein ligand.12,13 Such tive site. a tyrosyl-zinc interaction would preclude the large conformational change pro T h e R o l e o f Z i n c i n L i v e r posed on the basis of crystal structure A l c o h o l D ehydrogenase analysis. Liver alcohol dehydrogenase is a di In addition, the water molecule on the meric enzyme with two identical sub zinc atom has been shown by35Cl NMR units of molecular weight 40,000.5 Each investigations to interact with glutamic subunit contains two g atoms of zinc, only acid-270 though the metal may interact one of which is involved in catalytic ac with this residue directly.26 It should also tivity; the other is thought to stabilize be noted that the interaction of the en structure.7 X-ray crystallographic studies zyme with ester substrates is quite differ reveal two important differences be ent from that with peptides indicating tween these zinc ions. First, the active that there may be at least two alternative site zinc is liganded in a distorted tet catalytic mechanisms.2 Such studies em rahedral geometry to two cysteinyl sul- phasize the need for caution in ex furs and the imidazole group of a his trapolating from crystal structures to solu tidine. The fourth coordination position tion mechanisms. contains a water molecule. All four lig The significant feature of the zinc atom ands of the second zinc atom are cys is carboxypeptidase emerging from these teines. Second, the active site zinc is lo solution studies is its unusual coordina cated at the bottom of a hydrophobic poc tion state. Direct complexation with two ket about 25 Â from the protein surface residues implicated in the catalytic and can be approached from one direc mechanism suggests that this interaction tion by substrate and from a second by poises not only the metal but simultane the coenzyme nicotinamide-adenine di ously the organic components of the ac nucleotide (NAD). The structural zinc is tive site. Moreover, the grouping of located much closer to the enzyme sur amino acid side chains around the zinc face, and some 20 Â away from the active would effectively exclude water mole site. Its lack of a readily exchangeable cules from the substrate binding pocket ligand precludes its interaction with and perhaps, as a consequence, enhance chelating agents, coenzyme or substrate. 126 RIORDAN

E - Zn - H20 pulsory binding mechanism, addition of coenzyme induces a conformational •H NAD+ change in the protein that, among other things, results in the displacement of a NAD+ - E - Zn - OH" + H+ proton from the zinc-bound water mole cule. Substrate now binds to the zinc, + 4- RCH2 - OH presumably as the negatively charged al cohólate ion and displaces the hydroxyl NAD+ - E -Zn - "OCH2R + H20 ion. Hydride transfer to the coenzyme now occurs, and the resultant aldehyde + + dissociates from the enzyme to be re placed by water. This mechanism is NADH - E -Zn - OCHR analogous to that of the Meerwein- Pondorf-Oppenauer reaction. Both in + + volve hydride transfer to carboxyl com pounds and require participation of a NADH - E -Zn - H20 + RCHO strong Lewis acid.

R o l e o f Z i n c i n A s p a r t a t e T ranscarbamoylase E - Zn - H20 + NADH Aspartate transcarbamoylase from E.

F ig u r e 4. Schematic reaction mechanism for coli has been studied extensively be alcohol dehydrogenase catalysis (adapted from cause of interest in the mechanism of its reference 5). allosteric feedback regulation.11 The en What are the roles of the two distinct zyme can be dissociated into two types of classes of zinc in alcohol dehydrogenase? subunits, one which retains catalytic ac All attempts to remove either or both tivity and one which binds the regulator classes of zinc by treatment with molecule, CTP, but is inactive. It should chelators or dialysis at low pH have led to be noted that the regulatory subunits con the irreversible interaction of the en tain zinc, one g atom per 17,000 pro zyme. This implies a marked lability of tomeric weight. The role of zinc in the the metal-free protein and suggests that regulation of aspartate transcarbamoylase one pair of metal ions, presumably the is not entirely understood. Zinc seems to non-active site pair, serves to stabilize stabilize the tertiary structure of the reg structure. ulatory protomeric unit, promote its di- Since there is no evidence to support merization and is important for recon any other role for these metal ions and stitution of the native enzyme from its since it is unlikely that they do nothing, a separated subunits. Substitution of Hg2+ stabilizing function is the most obvious or Cd2+ for zinc gives a derivative with assignment at this time. However, x-ray properties nearly identical to those of the structural analysis provides no indication native enzyme. Zinc does not appear to that this part of the molecule is necessary be involved in binding the allosteric lig for structure stabilization and it has been and, CTP, to the regulatory subunit. suggested that the second zinc might T h e R o l e o f M e t a l s i n have evolutionary implications (5). A l k a l i n e P h o s p h a t a s e The role of the active site zinc is be lieved to be the mediation of elec- Escherichia coli alkaline phosphatase trophilic catalysis (figure 4). Following is a zinc metalloenzyme containing four g the well-known Theorell-Chance com atoms zinc per molecular weight of ROLE OF METALS IN ENZYME ACTIVITY 1 2 7

DNA REVERSE. 89,0003,25 As with alcohol dehyrogenase, * DNA* POLYMERASE^ ^ ^TRANSCRIPTASE each of the two identical subunits con tains two zinc atoms, one at the active site TRANSCRIPTION ___* RNA POLYMERASES♦ and one at another site. In addition the (r-RNA m-RNA) t-RNA enzyme, when isolated at neutral pH, TRANSLATION |,------A A -t-R N A ELONGATION FACTOR contains 1.3 g atoms of magnesium per I OTHER PROTEIN AMINO ACIDS mole.1,3 Magnesium alone does not acti Zn-E N ZY M E S PROTEASES AND vate the apoenzyme but increases the ac PEPTIDASES Zn-PROTEINS tivity of the enzyme containing two g atoms of zinc by about four-fold and that FIGURE 5. Zinc enzymes in nucleic acid and protein metabolism. of the four zinc enzyme by 20 percent. Hence, magnesium regulates the activ ment of microorganisms, plants, animals ity of alkaline phosphatase while zinc and, more recently, man.8,9,30 As evi serves, on the one hand, to stabilize struc denced by the few examples cited, the ture and, on the other, to participate in primary role of zinc would be to function the catalytic process. Magnesium inter in zinc metalloenzymes. However, it acts directly with the enzyme and does seems unlikely that disrupting the activ not seem to exert its regulatory role by ity of carboxypeptidase or alcohol dehy means of substrate binding. Studies with drogenase would have profound effects phosphatase containing cobalt instead of on growth. Moreover studies on the con zinc indicate that magnesium binding in sequences of zinc deficiency, particularly duces a change in the coordination in Euglena gracilis, indicated defects in geometry of the active site cobalt ions nucleic acid, protein synthesis and cellu and alters the relative affinities of cobalt lar division.8 or zinc for the catalytic, structural or reg Peptides, amino acids, nucleotides and ulatory sites.1 polyphosphate all accumulate under While it is not yet possible to define these conditions and the rate of incorpo more precisely the role of the three dif ration of [3H]-uridine into ribonucleic ferent classes of metal ions in alkaline acid (RNA) is markedly decreased. Cyto- phosphatase, this example illustrates fluorometric analysis of the metabolism quite well the emerging general princi of deoxyribonucleic acid (DNA) during ple. Metals in metalloenzymes can have the cell cycle of E. gracilis has revealed any one of three different roles,— that all of the biochemical processes es catalytic, structural or regulatory. The sential for cells to pass from G! into S, same metal, e.g., zinc, can have any one, from S into G2 and from G2 to mitosis re two or all three of these roles in the same quire zinc.9 It is now clear that zinc defi enzyme. Alternatively, different metals ciency disrupts these critical steps in the can fulfill these functions in a given en normal growth process because many of zyme. Hence, the analytical demonstra the important enzymes are zinc enzymes tion of the presence of a particular metal (figure 5). Thus, DNA polymerase, the species in an enzyme is not sufficient to various RNA polymerases, certain elon establish the specific role of that metal in gation factors and perhaps some amino biological function. acyl t-RNA syntheses all require zinc. Moreover, the RNA-dependent DNA T h e R o l e o f Z i n c i n N u c l e i c A c i d polymerases from avian, simian, feline and a n d P r o t e i n M e t a b o l i s m RD-114 tumor viruses have all been found to be zinc metalloenzymes.30 Such data Zinc has long been known to be essen extend the role of zinc in enzymes essen tial for the normal growth and develop- tial to normal nucleic acid metabolism to 128 RIORDAN others presumed to play a role in leukemic copper and zinc concentrations as well as processes. the hormonal influences on all of these, are yet to be defined. A complex inter play between storage proteins, carriers Conclusions and functional macromolecules would seem to underlie many of the biological Metalloenzymes are now well estab responses to changes in trace metal nutri lished entities in biochemistry, and their tion. Moreover, it should be noted that catalytic activities reflect the nutritional nucleic acids as well as proteins are importance of the corresponding essential known to bind many of the metals men minerals. At present it is recognized that tioned. Hence, some effects will not be copper and iron are especially important in due to changes in an enzyme activity but enzymes catalyzing oxidoreduction proc rather to the inability of these molecules esses while zinc, which can be a compo to exercise their assigned biological func nent of enzymes involved in a wide variety tions. On the basis of current knowledge, of reaction types, is critically associated it would seem that a good deal of progress with the fundamental steps of transcrip has been made in deciphering the role of tion and translation. The identification of metals in enzymes in elucidating their individual metalloenzymes is a relatively overall mode of action in biology. recent occurrence, particularly for zinc en zymes which are usually colorless. References

Two decades ago only three or four zinc 1. A n d e r s o n , R. A., K e n n e d y , F. S., and V a l - enzymes were known while 20 times that LEE, B. L.: The effect of Mg(II) on the spectral many are known today, and the number properties of Co(II) alkaline phosphatase. Biochemistry 25:3710-3716, 1976. increases steadily. Several dozen iron and 2. Au l d , D. S. and H o lm q u ist , B.: Carboxy- copper enzymes have been investigated peptidase A. Differences in the mechanisms of and, despite their more obvious charac ester and peptide hydrolysis. Biochemistry 13:4355-4361, 1974. teristics, new ones continue to be found. 3. Bosron, W. F., Ken n ed y , F. S., and Va l l ee , F ewer examples of cobalt, manganese and B. L.: Zinc and magnesium content of alkaline selenium enzymes are presently known; phosphatase from Escherichia coli. Biochemis try 24:2275-2282, 1975. however, based on experience with other 4. B r a d s h a w , R. A., E r ic c s o n , L. H., W a l s h , K. metals, this situation is expected to change A., and N e u r a t h , H.: The amino acid se with time as well. quence of bovine carboxypeptidase A. Proc. Nat. Acad. Sei. 63:1389-1394, 1969. As the number of known metalloen 5. B r ä n d e n , C. I., JÖ r n v a l l , H., E k l u n d , H., and F u r u g r e n , B.: Alcohol dehydrogenases. zymes increases, the metabolic and The Enzymes, vol. XI. Boyer, P. D., ed. New physiologic consequences of metal defi York, Academic Press, pp. 103-190, 1975. ciency begin to be understood. The 6. D ix o n , N. E., Ga z z o la , C., Bla k eley , R. L., and ZERNER, B.: Jack bean urease (EC3.5.1.0). growth retardation and teratogenic ef A metalloenzyme. A simple biological role for fects of zinc deficiency or the weakening nickel? J. Amer. Chem. Soc. 97:4131-4133, of elastic tissue owing to copper defi 1975. 7. D r u m , D . E. and V a l l e e , B. L.: Differential ciency, can be traced, at least in part, to chemical reactivities of zinc in horse liver al specific enzymes or groups of enzymes. cohol dehydrogenase. Biochemistry 9:4078- However, many aspects of trace metal 4086, 1970. 8. Falchuk, K. F., Faw cett, D. W., and Val metabolism remain unknown. The rela l e e , B. L.: Role of zinc in cell division of tionships between trace metals, such as Euglena gracilis. J. Cell. Sei. 17:57-78, 1975. the reciprocal alterations in zinc and 9. F a l c h u k , K. F ., Kr is h a n , A., and V a l l e e , B. L.: DNA distribution in the cell cycle of Eu copper concentrations in blood serum, glena gracilis. Cytofluorometry of zinc defi the effects of manganese and iron on cient cells. Biochemistry 24:3439-3444, 1975. ROLE OF METALS IN ENZYME ACTIVITY 1 2 9

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