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Role of metalloenzymes in biological system.
Certain metals have long been recognized to have important biological functions primarily as a
consequence of nutritional investigations. Thus, the absence of a specific, essential metal from
the diet of an organism invariably leads to a deficiency state characterized by metabolic
abnormalities with altered or retarded growth (Anderson et al., 1976). Because such metals are
usually present in tissues in very small amounts it was reasonable to suspect that they might play
a catalytic role, perhaps participating in enzymatic reactions. The actual discovery of
metalloenzymes, however, required the availability of accurate, sensitive, analytical
methodology. As a consequence, the unequivocal demonstration of a role for metals in enzyme
action is of relatively recent vintage. At present, reliable measurements of small concentrations
of metals present in tissues, cells, subcellular particles, body fluids and bio macromolecules can
be performed by colorimetry, fluorimetry, polarography, emission spectrometry with spark,
flame or plasma excitation sources, x-ray and atomic fluorescence, atomic absorption and
neutron activation analysis, among other methods. Metals that have been detected by such
techniques and currently known to be components of metalloenzymes include cobalt, copper,
iron, manganese, molybdenum, nickel, selenium and zinc. Aside from its role in vitamin B12,
cobalt, to date, has been found to be a component of but one enzyme, the component of but one
enzyme, the biotin-dependent, zinc-containing oxaloacetate trans carboxylase of
Propionibacterium shermanii (Auld and Holmquist, 1974). Copper is present in a large number
of enzymes that catalyze oxidoreduction reactions such as tyrosinase, lysyl oxidase and
cytochrome oxidase. Iron is also found primarily in enzymes that participate in oxidoreduction
reactions; in addition, it plays a major role in oxygen transport.18 Manganese has been identified
as a component of pyruvate carboxylase from chicken liver and is present in Escherichia coli
superoxide dismutase.15 It also serves as an activator for many metal-activated enzymes;
however, in most of these cases, magnesium and other divalent cations can fulfill the same
function. Molybdenum is found most frequently in flavin-dependent enzymes, usually in
conjunction with non-heme iron and acid-labile sulfur. A typical example is xanthine oxidase. A
molybdoheme protein, sulfite oxidase, has been described as well as a molybdoferrodoxin, a
component of the nitrogenase system of nitrogen-fixing bacteria Azotobacter vinelandii and
Clostridium pasteurianum-15 Nickel has been found to be present in urease 50 years after the
enzyme was first crystallized.8 Selenium, which has been recognized as an essential nutrient for
more than a dozen years, has recently been shown to be a component of an enzyme, glutathione
peroxidase from erythrocytes, the first example of a selenoenzyme. Zinc enzymes are among the
most common of the metalloenzymes numbering over 70 and representing each of the six
categories of enzymes designated by the International Union on Biochemistry (IUB) commission
on enzymes. Zinc metalloenzymes exhibit perhaps the greatest diversity both of catalytic
function and of the role played by the metal atom (Bradshaw et al., 1969). The metal is present
in several dehydrogenases, aldolases, peptidases and phosphatases. Zinc enzymes participate in
carbohydrate, lipid, protein and nucleic acid synthesis or degradation. Several examples of zinc
enzymes will be cited to illustrate the role metals in metalloenzymes and the general importance
of zinc to metabolism. Other metals such as sodium, potassium, calcium and magnesium can also
assist in the action of enzymes. With these, the mode of metal-enzyme interaction is complex
and often difficult to establish. Still other metals, such as chromium, vanadium and tin, have
been shown to be either essential for growth in certain species or components of biological
macromolecules. However, their relationship to enzyme mechanisms has not been established.
Enzymes affected by metal ions have been operationally defined as either metalloenzymes or
metal-enzyme complexes. A metalloenzymes contains a firmly bound, stoichiometric quantity of
a metal metal as an integral part of the protein molecule. Removal of the metal by treatment with
chelating agents, for example, abolishes catalytic activity (Bränden et al., 1975).
The Role of Zinc in Metalloenzymes
The role of zinc in carboxypeptidaase.
Carboxypeptidase A is a classic zinc metalloenzyme.31 It contains one g atom of zinc per
molecular weight (34,500). Removal of the metal atom either by dialysis at low pH or by
treatment with chelating agents gives a totally inactive apoenzyme. Activity can be restored by
re-addition of zinc or one of a number of other divalent metal ions. 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 peptidase activity of cadmium carboxypeptidase is a function
of the particular peptide 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 carboxypeptidases are
essentially inactive as peptidase. A comparison of the kinetic parameters for the zinc, cobalt,
manganese and cadmium enzyme-catalyzed hydrolysis of benzoyl-glycyl-glycyl-Lphenylalanine
xrayreveal a range of kcat values from 6000 min”1 for the cobalt enzyme to 43 min-1 for the
cadmium enzyme. The Km values, on the other hand, are almost totally independent of the
particular metal present. Thus, it would appear that the primary role of the metal is to function in
the catalytic process and that it has little to do with substrate binding. This is consistent with
previous studies showing that peptide substrates bind to apocarboxypeptidase and prevent the
reassociation of the metal-free protein with zinc (Bosron et al., 1975).
The role of zinc in liver alcohol dehydrogenase.
Liver alcohol dehydrogenase is a dimeric enzyme with two identical subunits of molecular
weight 40,000.5 Each subunit contains two g atoms of zinc, only one of which is involved in
catalytic activity; the other is thought to stabilize structure.7 X-ray crystallographic studies
reveal two important differences between these zinc ions. First, the active site zinc is liganded in
a distorted tetrahedral geometry to two cysteinyl sulfurs and the imidazole group of a histidine.
The fourth coordination position contains a water molecule. All four ligands of the second zinc
atom are cysteines. Second, the active site zinc is located at the bottom of a hydrophobic pocket
about 25 Â from the protein surface and can be approached from one direction by substrate and
from a second by the coenzyme nicotinamide-adenine dinucleotide (NAD). The structural zinc is
located much closer to the enzyme surface, and some 20 Â away from the active site. Its lack of
a readily exchangeable ligand precludes its interaction with chelating agents, coenzyme or
substrate (Dixon, et al., 1975).
Role of zinc in aspartate transcarbamoylase.
Aspartate transcarbamoylase from E. coli has been studied extensively because of interest in the
mechanism of its allosteric feedback regulation. The enzyme can be dissociated into two types of
subunits, one which retains catalytic activity and one which binds the regulator molecule, CTP,
but is inactive. It should be noted that the regulatory subunits contain zinc, one g atom per
17,000 protomeric weight. The role of zinc in the regulation of aspartate transcarbamoylase is
not entirely understood. Zinc seems to stabilize the tertiary structure of the regulatory protomeric
unit, promote its dimerization and is important for reconstitution of the native enzyme from its
separated subunits. Substitution of Hg2+ or Cd2+ for zinc gives a derivative with properties
nearly identical to those of the native enzyme. Zinc does not appear to be involved in binding the
allosteric ligand, CTP, to the regulatory subunit (Vallee, B.L 1955).
The role of metals in alkaline phosphatase.
Escherichia coli alkaline phosphatase is a zinc metalloenzyme containing four g atoms zinc per
molecular weight of 89,0003. As with alcohol dehyrogenase, each of the two identical subunits
contains two zinc atoms, one at the active site and one at another site. In addition, the enzyme,
when isolated at neutral pH, contains 1.3 g atoms of magnesium per mole. Magnesium alone
does not activate the apoenzyme but increases the activity of the enzyme containing two g atoms
of zinc by about four-fold and that of the four zinc enzyme by 20 percent. Hence, magnesium
regulates the activity of alkaline phosphatase while zinc serves, on the one hand, to stabilize
structure and, on the other, to participate in the catalytic process. Magnesium interacts directly
with the enzyme and does not seem to exert its regulatory role by means of substrate binding.
Studies with phosphatase containing cobalt instead of zinc indicate that magnesium binding
induces a change in the coordination geometry of the active site cobalt ions and alters the relative
affinities of cobalt or zinc for the catalytic, structural or regulatory sites.
The role of zinc in nucleic acid and protein metabolism.
Zinc has long been known to be essential for the normal growth and development of
microorganisms, plants, animals and, more recently, man. As evidenced by the few examples
cited, the primary role of zinc would be to function in zinc metalloenzymes. However, it seems
unlikely that disrupting the activity of carboxypeptidase or alcohol dehydrogenase would have
profound effects on growth. Moreover, studies on the consequences of zinc deficiency,
particularly in Euglena gracilis, indicated defects in nucleic acid, protein synthesis and cellular
division. Peptides, amino acids, nucleotides and polyphosphate all accumulate under these
conditions and the rate of incorporation of [3H]-uridine into ribonucleic acid (RNA) is markedly
decreased. Cytofluorometric analysis of the metabolism of deoxyribonucleic acid (DNA) during
the cell cycle of E. gracilis has revealed that all of the biochemical processes essential for cells to
pass from G! into S, from S into G2 and from G2 to mitosis require zinc.9 It is now clear that
zinc deficiency disrupts these critical steps in the normal growth process because many of the
important enzymes are zinc enzymes. Thus, DNA polymerase, the various RNA polymerases,
certain elongation factors and perhaps some amino acyl t-RNA syntheses all require zinc.
Moreover, the RNA-dependent DNA polymerases from avian, simian, feline and RD-114 tumor
viruses have all been found to be zinc metalloenzymes. Such data extend the role of zinc in
enzymes essential to normal nucleic acid metabolism to others presumed to play a role in
leukemic processes (Vallee, B.L 1973).
References
Anderson, R.A., Kennedy, F.S., and Vallee, B.L (1976). The effect of Mg(II) on the spectral
properties of Co(II) alkaline phosphatase. Biochemistry (25):3710-3716.
Auld, D.S. and Holmquist, B (1974). Carboxypeptidase A. Differences in the mechanisms of
ester and peptide hydrolysis. Biochemistry (13):4355-4361.
Bosron, W.F., Kennedy, F.S., and Vallee, B. L (1975). Zinc and magnesium content of alkaline
phosphatase from Escherichia coli. Biochemistry (24):2275-2282.
Bradshaw, R.A., Ericcson, L.H., Walsh, K.A., and Neurath, H. (1969). The amino acid sequence
of bovine carboxypeptidase. Amino acid (63):1389-1394.
Bränden, C.I., JÖrnvall, H., Eklund, H., and Furugren, B (1975). Alcohol dehydrogenases. The
Enzymes, volume XI. Boyer, P.D., ed. New York, Academic Press, pp. 103-190, 1975.
Dixon, N.E., Gazzola, C., Blakeley, R L., and ZERNER, B.: Jack bean urease (1975). A
metalloenzymes. A simple biological role for nickel? J. American Chemistry Society (97):41314133.
Vallee, B.L (1955).: Zinc and metalloenzymes. Advance Protein Chemistry (10):317-334.
Vallee, B L. (1973). Cobalt substituted zinc metalloenzymes. Metal Ions in Biological Systems.
Dhar, S. K., ed. New York, Plenum, pp. 1-12.