ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 2 Copyright © 1977, Institute for Clinical Science The Role of Metals in Enzyme Activity* 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 metalloen- zymes 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 metalloen- zymes, 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.