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. Carboxypeptidase 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
Certain metals have long been recog­
nized to have important biological func­
tions primarily as a consequence of nu­
tritional investigations.14,15,22 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. Because such metals
are usually present in tissues in very
small amounts it was reasonable to sus­
pect that they might play a catalytic role,
perhaps participating in enzymatic reac­
tions. The actual discovery of metalloenzymes, however, required the availability
of accurate, sensitive, analytical method­
ology. As a consequence, the unequivo­
cal demonstration of a role for metals in
enzyme action is of relatively recent vin­
tage.
119
At present, reliable measurements of
small concentrations of metals present in
tissues, cells, subcellular particles, body
fluids and biomacromolecules 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, molyb­
denum, nickel, selenium and zinc (table
I).
Aside from its role in vitamin B 12,
cobalt, to date, has been found to be a
* Supported by Grant-in-Aid GM-15003 from the
National Institutes of Health of the Department of
Health, Education and Welfare.
RIORDAN
12 0
TABLE I
Metals Present in Naturally Occurring Metalloenzymes
Enzyme Function
Metal
Transcarboxylation
Oxidoreduction
Oxidoreduction
Various
Oxidoreduction
Urease
Peroxidase
Various
Cobalt
Copper
Iron
Manganese
Molybdenum
Nickel
Selenium
Zinc
component of but one enzyme, the
biotin-dependent,
zinc-containing
oxaloacetate transcarboxylase of Pro­
pionibacterium shermanii.19 Copper is
present in a large number of enzymes
that catalyze oxidoreduction reactions
such as tyrosinase, lysyl oxidase and
cytochrome oxidase.20 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 com­
ponent of pyruvate carboxylase from
chicken liver and is present in Es­
cherichia 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 func­
tion.
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 pro­
tein, sulfite oxidase, has been described
TABLE
II
Currently Known Zinc Metalloenzymes
I n t e r n a t io n a l Union
o f B io ch em istry System
E.C.l
E.C.2
E.C.3
E.C.4
E.C.5
E.C.6
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Number
7
8
23
19
1
1
Example
Alcohol dehydrogenases
DN A polymerase
Carboxypeptidase
6-ALA dehydratase
Mannose-P isomerase
Pyruvate carboxylase
as well as a molybdoferrodoxin, a com­
ponent 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 en­
zyme, glutathione peroxidase from eryth­
rocytes, the first example of a selenoenzyme.10
Zinc enzymes are among the most
common of the metalloenzymes number­
ing over 70 and representing each of the
six categories of enzymes designated by
the International Union on Biochemistry
(IUB) commission on enzymes (table II).
Zinc metalloenzymes exhibit perhaps the
greatest diversity both of catalytic func­
tion and of the role played by the metal
atom.15,21,22’23,27'28,30 The metal is present
in several dehydrogenases, aldolases,
peptidases and phosphatases. Zinc en­
zymes participate in carbohydrate, lipid,
protein and nucleic acid synthesis or de­
gradation. Several examples of zinc en­
zymes will be cited to illustrate the role
metals in metalloenzymes and the gen­
eral importance of zinc to metabolism.
Other metals such as sodium, potas­
sium, calcium and magnesium can also
assist in the action of enzymes. With th­
ese, the mode of metal-enzyme interac­
tion is complex and often difficult to es­
tablish. Still other metals, such as
chromium, vanadium and tin, have been
shown to be either essential for growth in
certain species or components of biologi­
cal macromolecules. However, their rela­
tionship to enzyme mechanisms has not
been established.
Enzymes affected by metal ions have
been operationally defined as either
metalloenzymes or metal-enzyme com­
plexes.28 A metalloenzyme contains a
firmly bound, stoichiometric quantity of a
ROLE OF METALS IN ENZYME ACTIVITY
metal as an integral part of the protein
molecule. Removal of the metal by treat­
ment with chelating agents, for example,
abolishes catalytic activity. In instances
where the resultant apoenzyme is structually stable, restoration of the metal can
regenerate full biological function. In
contrast, metal-enzyme complexes are
more loosely associated, the criterion for
association being metal activation of
catalysis. The metal ion is frequently not
an integral part of the molecule when iso­
lated, and the enzyme may exhibit partial
activity in the absence of the metal ion.
Obviously, the difference between these
two classes of metal-enzyme systems de­
pends on the magnitude of the metalprotein stability constant which can be a
function of the metal atom as well as en­
vironmental conditions such as pH, buf­
fer and ionic strength. The metalloenzymes are better suited for elucidation of
the metal protein interaction and for ex­
trapolating such information to the un­
derstanding of enzymic mechanisms.
Moreover, they lend themselves more
readily to a definite assessment of the
physiological role of the metal. Metalenzyme complexes, however, have been
of great theoretical importance in the un­
derstanding of catalytic phenomena and
general mechanisms of catalysis by
metalloenzymes.
At present some 2,000 or more different
enzymes have been isolated and charac­
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
greater for it has been pointed out that
“There probably does not exist a single
enzyme-catalyzed reaction in which
either enzyme, substrate, product, or a
combination of these is not influenced in
a very direct and highly specific manner
by the precise nature of the inorganic
ions which surround and modify it” .17
This paper will be concerned primarily
121
with the role of metals in metalloen­
zymes and will not attempt to cover the
interesting but voluminous literature de­
aling with metal-enzyme complexes.
The Interaction of Metal Ions
With Enzymes
A number of schemes have been pres­
ented24 to describe the types of interac­
tions that can occur between metals, en­
zyme proteins and substrate (or inhibitor)
(figure 1). The first of these represents an
interaction between the substrate and the
metal ion to form a complex that acts as
the true substrate. Substrate-metal complexation can occur prior or subsequent
to the formation of the enzyme-substrate
complex. This type of behavior is typi­
cally observed with metal-activated en­
zymes. The second scheme indicates that
the metal first binds to the protein and
then serves as a site of interaction with
substrate. In this instance, the metal can
function either as a binding site, as a
component of the catalytic apparatus of
the enzyme or both.
An example of both such possibilities is
given by the role of zinc in carboxypeptidase A. Here the zinc atom is believed
to interact with a peptide substrate via
the carbonyl oxygen atom of its terminal
M + S = MS
E + MS = EMS
2) E
+ M = EM
} EM + S = EMS
3, E
} ME
+ M = ME
+ S = MES
F i g u r e 1. Interactions between metal (M), en­
zyme (E) and substrate (S).
122
RIORDAN
peptide bond, i.e., the one that is suscep­
tible to hydrolysis. However, even
though some kind of metal-substrate
bond may be formed, the metal does not
appear to be essential for peptide sub­
strate binding. Peptides bind to the
metal-free apoenzyme as well as they do
to the metalloenzyme, even though they
are not hydrolyzed.2 Thus, for peptide
substrates the metal presumably serves
as a catalytic site. On the other hand,
ester substrates of carboxypeptidase do
not bind to the apoenzyme. It has been
proposed that differences in the mode of
interaction between substrate and metal
account for the numerous kinetic differ­
ences that have been observed for car­
boxypeptidase acting on ester and pep­
tide substrates, respectively.2
A third scheme would have the metal
acting at a site on the enzyme remote
from the active site. In such instances,
the metal could either serve to maintain
protein structure and only influence
catalytic activity indirectly or else it
could regulate activity by stabilizing
more or less active conformations of the
protein. The latter situation would more
likely pertain for metal-activated en­
zymes where the metal-protein interac­
tion is more readily controlled by manip­
ulation of the ambient metal ion concen­
tration. It should be emphasized that
these schemes are not all mutually exclu­
sive and that some metalloenzymes are
known to contain functionally different
classes of metal ions.
The Role of Metals in the Mechanism
of Catalysis
Iron, copper and molybdenum are most
commonly encountered in enzymes
catalyzing oxidoreduction reactions. In
the majority of cases, the metal ion par­
ticipates directly in the electron transfer
process and undergoes a cyclic change in
oxidation state. Oftentimes the free metal
is capable of catalysis by itself as with the
iron-promoted decomposition of hydro­
gen peroxide although in this case
catalase is at least a million times more
effective than iron alone. Thus, the pro­
tein component of a metalloenzyme con­
tributes many of the critical aspects of the
catalytic mechanism.
Zinc, on the other hand, does not
undergo a change in oxidation state dur­
ing enzymatic catalysis even though it
participates in oxidoreduction reactions,
e.g., as a component of alcohol dehydro­
genase. The zinc cation has a stable, d 10
electronic configuration and has little
tendency to accept or to donate single
electrons. Instead, it serves as a Lewis
acid interacting with electronegative
donors to increase the polarity of chemi­
cal bonds and thus promote the transfer
of atoms or groups. Substitution reactions
of simple metal chelates generally pro­
ceed via intermediates with an open
coordination position or a distorted coor­
dination sphere. Zinc (and also cobalt)
can readily accept a distorted geometry
and, hence, would appear to be well
suited to participate in substitution reac­
tions as, for instance, in carbonic anhydrase, carboxypeptidase and alkaline
phosphatase.
Entasis and Metalloenzyme Active Sites
What then is the role of the protein in
the mechanism of action of a metalloen­
zyme? As indicated for catalase, it makes
a major contribution to the enhancement
of reaction rate. It creates a proper bind­
ing locus to ensure substrate specificity.
It juxtaposes catalytic residues in the
precise orientation with respect to the
susceptible reaction centers of the sub­
strate. It provides a suitably balanced
hydrophobic-hydrophilic environment
and serves to collect all the participating
species in reactions between several
molecules. It also provides liganding
groups for binding the metal. The
number, nature, orientation and im­
ROLE OF METALS IN ENZYME ACTIVITY
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
states and approximating that of the
plausible transition state for the reaction
in which it is involved. Vallee and Wil­
liams have called this an entatic state im­
plying a state of tension at the active site
of the enzyme.32,33 It has been defined by
them as “the existence in the enzyme of
an area with energy, closer to that of a
unimolecular transition state than to that
of a conventional stable molecule,
thereby constituting an energetically
poised domain.”
Spectral Properties of Metalloenzymes
On the basis of entasis, the
physicochemical properties of metals in
metalloenzymes might be expected to di­
ffer from those observed for the same
metals present in well-defined model
coordination complexes. Both the absorp­
tion and EPR* spectra of copper and
nonheme iron enzymes are unusual
when compared to those of simple copper
and iron complexes.32,32 In particular the
so-called copper blue enzymes exhibit
absorption bands that differ strikingly in
intensity and fine structure from those of
non-catalytic copper proteins.
The zinc ion has neither intrinsic color
nor unpaired electrons hence its
* Electron spin resonance.
123
FIGURE 2. Visible absorption spectra of E. coli
alkaline phosphatase containing 4 g atoms of cobalt
per mole of protein compared with spectra of cobalt
model complexes.
physicochemical properties are difficult
to assess. However, in virtually every
zinc metalloenzyme where it has been at­
tempted, the zinc ion can be replaced by
cobalt to form an enzymically active de­
rivative with a visible absorption spec­
trum.29 The resulting spectra differ signif­
icantly from the spectra of model cobalt
(II) complexes as shown for alkaline
phosphatase in figure 2. Moreover, altera­
tion of the coordination sphere by the ad­
dition of another ligand such as an inTABLE
III
Activities of Metallocarboxy-Peptidases
Activity
Peptidase*
Metal
Apo
Zinc
Cobalt
Nickel
Manganese
Cadmium
Mercury
Rhodium
Lead
Copper
(v/vzinc X 100)
(v' vzinc x 100>
0
100
200
47
27
0
0
0
0
0
Esteraset
0
100
114
43
156
143
86
71
57
0
*0.02 M benzyloxycarbonylglycyl-L-phenylalanine,
pH 7.5, 0° C.
tO . 01 M benzoylglycyl-DL-phenyllactate, p H 7.5,
25° C.
riordan
124
F i g u r e 3. Schematic representation of the
mechanism of peptide hydrolysis catalyzed by carboxypeptidase A.
hibitor anion converts the irregular spec­
trum to one closely resembling a regular
tetrahedral cobalt ion. It is important to
note that these properties can be ob­
served in the absence of substrate. While
it has not been possible to interpret these
unusual properties of metals in metalloenzymes in terms of precise geometries
and, ultimately, mechanisms of enzyme
action, nevertheless they are quite con­
sistent with views on the entatic nature of
active sites.
The Role of Zinc in Metalloenzymes
The Ro le
of
Z in c
in
C a r b o x y p e p t id a s e
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.22 23 Activity can be restored
by readdition of zinc or one of a number
of other divalent metal ions (table III).
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 carboxypeptidases 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-Lphenylalanine (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 Kmvalues, on the other hand,
are almost totally independent of the par­
ticular metal present. Thus, it would ap­
pear that the primary role of the metal is
to function in the catalytic process and
that it has little to do with substrate bind­
ing. This is consistent with previous
studies showing that peptide substrates
bind to apocarboxypeptidase and prevent
the reassociation of the metal-free protein
with zinc.
X-ray analysis of carboxypeptidase
crystals together with amino acid se­
quence information has identified three
protein ligands to the zinc.416 They are
glutamic acid-72, histidine-69 and
histidine-196. Both histidyl residues are
held in position by hydrogen bonding to
carboxyl side chains. In the crystalline
state, a fourth coordination site is oc­
cupied by water. Diffusion of the very
slowly hydrolyzed dipeptide, glycyl-Ltyrosine, into the crystals gives an
enzyme-dipeptide complex. The car­
bonyl oxygen atom of the substrate’s pep­
tide bond is thought to displace the coor­
dinated water atom and interact directly
with the zinc. This polarizes the carbonyl
bond and promotes an attack by the car­
bonyl group of glutamic acid-270 at the
ROLE OF METALS IN ENZYME ACTIVITY
carbonyl carbon atom either directly or
through a water molecule (figure 3). A
key feature of the catalytic mechanism
porposed by the x-ray crystallographers is
a 12A movement of tyrosine-248, a resi­
due thought to serve as a proton donor to
the susceptible peptide nitrogen atom.16
Several conclusions drawn from
studies carried out with carboxypeptidase in solution differ significantly from
those derived from x-ray analysis. Using a
chemically modified derivative of carboxypeptidase in which tyrosine-248 was
coupled with diazotized arsanilic acid,
Johansen and Vallee demonstrated that
the azotyrosyl residue interacts directly
with the zinc ion thus constituting a
fourth and a fifth protein ligand.12,13 Such
a tyrosyl-zinc interaction would preclude
the large conformational change pro­
posed on the basis of crystal structure
analysis.
In addition, the water molecule on the
zinc atom has been shown by 35C l NMR
investigations to interact with glutamic
acid-270 though the metal may interact
with this residue directly.26 It should also
be noted that the interaction of the en­
zyme with ester substrates is quite differ­
ent from that with peptides indicating
that there may be at least two alternative
catalytic mechanisms.2 Such studies em­
phasize the need for caution in ex­
trapolating from crystal structures to solu­
tion mechanisms.
The significant feature of the zinc atom
is carboxypeptidase emerging from these
solution studies is its unusual coordina­
tion state. Direct complexation with two
residues implicated in the catalytic
mechanism suggests that this interaction
poises not only the metal but simultane­
ously the organic components of the ac­
tive site. Moreover, the grouping of
amino acid side chains around the zinc
would effectively exclude water mole­
cules from the substrate binding pocket
and perhaps, as a consequence, enhance
125
TABLE
IV
Metallocarboxypeptidase - Catalyzed Hydrolysis
of Benzy1-glycylglycyl-L-phenylalanine
k
M etal
Cobalt
Zinc
Manganese
Cadmium
c a t (mm
-1
)
6,000
1,200
230
41
Km
(mM 1)
1.5
1.0
2.8
1.3
the catalytic properties of both glutamic
acid-270 and tyrosine-248. The most im­
portant thing, however, is that zinc ion is
central to the overall process of hyd­
rolysis. Its major function is to be able to
undergo ligand exchange when triggered
by the entrance of substrate into the ac­
tive site.
The Role
Al c o h
ol
of
D
Z in c
in
L iv e r
ehydrogenase
Liver alcohol dehydrogenase is a di­
meric enzyme with two identical sub­
units of molecular weight 40,000.5 Each
subunit contains two g atoms of zinc, only
one of which is involved in catalytic ac­
tivity; the other is thought to stabilize
structure.7 X-ray crystallographic studies
reveal two important differences be­
tween these zinc ions. First, the active
site zinc is liganded in a distorted tet­
rahedral geometry to two cysteinyl sulfurs and the imidazole group of a his­
tidine. The fourth coordination position
contains a water molecule. All four lig­
ands of the second zinc atom are cys­
teines. Second, the active site zinc is lo­
cated at the bottom of a hydrophobic poc­
ket about 25 Â from the protein surface
and can be approached from one direc­
tion by substrate and from a second by
the coenzyme nicotinamide-adenine di­
nucleotide (NAD). The structural zinc is
located much closer to the enzyme sur­
face, 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.
RIORDAN
126
E - Zn - H20
NAD+
NAD+
NADH
NADH
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
- 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
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
E-Zn - OCHR
analogous to that of the MeerweinPondorf-Oppenauer reaction. Both in­
++
volve hydride transfer to carboxyl com­
pounds and require participation of a
E-Zn - H20 +
RCHO
strong Lewis acid.
Role
E - Zn - H20 +
NADH
F i g u r e 4.
Schematic reaction mechanism for
alcohol dehydrogenase catalysis (adapted from
reference 5).
What are the roles of the two distinct
classes of zinc in alcohol dehydrogenase?
All attempts to remove either or both
classes of zinc by treatment with
chelators or dialysis at low pH have led to
the irreversible interaction of the en­
zyme. This implies a marked lability of
the metal-free protein and suggests that
one pair of metal ions, presumably the
non-active site pair, serves to stabilize
structure.
Since there is no evidence to support
any other role for these metal ions and
since it is unlikely that they do nothing, a
stabilizing function is the most obvious
assignment at this time. However, x-ray
structural analysis provides no indication
that this part of the molecule is necessary
for structure stabilization and it has been
suggested that the second zinc might
have evolutionary implications (5).
The role of the active site zinc is be­
lieved to be the mediation of electrophilic catalysis (figure 4). Following
the well-known Theorell-Chance com­
of
Z in c
in
A spartate
Tr a n sc a r b a m o y la se
Aspartate transcarbamoylase from E.
coli has been studied extensively be­
cause of interest in the mechanism of its
allosteric feedback regulation.11 The en­
zyme can be dissociated into two types of
subunits, one which retains catalytic ac­
tivity and one which binds the regulator
molecule, CTP, but is inactive. It should
be noted that the regulatory subunits con­
tain zinc, one g atom per 17,000 pro­
tomeric weight. The role of zinc in the
regulation of aspartate transcarbamoylase
is not entirely understood. Zinc seems to
stabilize the tertiary structure of the reg­
ulatory protomeric unit, promote its dimerization and is important for recon­
stitution 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 lig­
and, CTP, to the regulatory subunit.
The Role
of
M
e t a l s in
A l k a l in e P h o s p h a t a s e
Escherichia coli alkaline phosphatase
is a zinc metalloenzyme containing four g
atoms zinc per molecular weight of
ROLE OF METALS IN ENZYME ACTIVITY
89,0003,25 As with alcohol dehyrogenase,
each of the two identical subunits con­
tains 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.1,3 Magnesium alone does not acti­
vate the apoenzyme but increases the ac­
tivity 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 activ­
ity of alkaline phosphatase while zinc
serves, on the one hand, to stabilize struc­
ture and, on the other, to participate in
the catalytic process. Magnesium inter­
acts 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 in­
duces 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 reg­
ulatory sites.1
While it is not yet possible to define
more precisely the role of the three dif­
ferent classes of metal ions in alkaline
phosphatase, this example illustrates
quite well the emerging general princi­
ple. Metals in metalloenzymes can have
any one of three different roles,—
catalytic, structural or regulatory. The
same metal, e.g., zinc, can have any one,
two or all three of these roles in the same
enzyme. Alternatively, different metals
can fulfill these functions in a given en­
zyme. Hence, the analytical demonstra­
tion of the presence of a particular metal
species in an enzyme is not sufficient to
establish the specific role of that metal in
biological function.
The Role
and
of
Z
P r o t e in M
in c in
N u c l e ic A c id
e t a b o l is m
Zinc has long been known to be essen­
tial for the normal growth and develop-
127
DNA
TRANSCRIPTION
REVERSE.
^TRANSCRIPTASE
___* ♦
RNA POLYMERASES
(r-R N A
TRANSLATION
^
*DNA*
POLYM ERASE^
m -R N A )
t-R N A
|,------- A A -t-R N A
ELONGATION FACTOR
OTHER
Z n -E N Z Y M E S
AND
Zn-PROTEINS
I
PROTEIN
AMINO ACIDS
PROTEASES
PEPTIDASES
FIGURE 5. Zinc enzymes in nucleic acid and
protein metabolism.
ment of microorganisms, plants, animals
and, more recently, man .8,9,30 As evi­
denced by the few examples cited, the
primary role of zinc would be to function
in zinc metalloenzymes. However, it
seems unlikely that disrupting the activ­
ity of carboxypeptidase or alcohol dehy­
drogenase would have profound effects
on growth. Moreover studies on the con­
sequences of zinc deficiency, particularly
in Euglena gracilis, indicated defects in
nucleic acid, protein synthesis and cellu­
lar division.8
Peptides, amino acids, nucleotides and
polyphosphate all accumulate under
these conditions and the rate of incorpo­
ration 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 es­
sential for cells to pass from G! into S,
from S into G 2 and from G 2 to mitosis re­
quire zinc.9 It is now clear that zinc defi­
ciency disrupts these critical steps in the
normal growth process because many of
the important enzymes are zinc enzymes
(figure 5). Thus, DNA polymerase, the
various RNA polymerases, certain elon­
gation 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.30 Such data
extend the role of zinc in enzymes essen­
tial to normal nucleic acid metabolism to
128
RIORDAN
others presumed to play a role in leukemic
processes.
Conclusions
Metalloenzymes are now well estab­
lished entities in biochemistry, and their
catalytic activities reflect the nutritional
importance of the corresponding essential
minerals. At present it is recognized that
copper and iron are especially important in
enzymes catalyzing oxidoreduction proc­
esses while zinc, which can be a compo­
nent of enzymes involved in a wide variety
of reaction types, is critically associated
with the fundamental steps of transcrip­
tion and translation. The identification of
individual metalloenzymes is a relatively
recent occurrence, particularly for zinc en­
zymes which are usually colorless.
Two decades ago only three or four zinc
enzymes were known while 20 times that
many are known today, and the number
increases steadily. Several dozen iron and
copper enzymes have been investigated
and, despite their more obvious charac­
teristics, new ones continue to be found.
F ewer examples of cobalt, manganese and
selenium enzymes are presently known;
however, based on experience with other
metals, this situation is expected to change
with time as well.
As the number of known metalloen­
zymes increases, the metabolic and
physiologic consequences of metal defi­
ciency begin to be understood. The
growth retardation and teratogenic ef­
fects of zinc deficiency or the weakening
of elastic tissue owing to copper defi­
ciency, can be traced, at least in part, to
specific enzymes or groups of enzymes.
However, many aspects of trace metal
metabolism remain unknown. The rela­
tionships between trace metals, such as
the reciprocal alterations in zinc and
copper concentrations in blood serum,
the effects of manganese and iron on
copper and zinc concentrations as well as
the hormonal influences on all of these,
are yet to be defined. A complex inter­
play between storage proteins, carriers
and functional macromolecules would
seem to underlie many of the biological
responses to changes in trace metal nutri­
tion. Moreover, it should be noted that
nucleic acids as well as proteins are
known to bind many of the metals men­
tioned. Hence, some effects will not be
due to changes in an enzyme activity but
rather to the inability of these molecules
to exercise their assigned biological func­
tions. On the basis of current knowledge,
it would seem that a good deal of progress
has been made in deciphering the role of
metals in enzymes in elucidating their
overall mode of action in biology.
References
1. A n d e r s o n , R. A., K e n n e d y , F . S., and V a l LEE, B. L.: The effect of Mg(II) on the spectral
properties o f C o (II) alkaline phosphatase.
Biochemistry 25:3710-3716, 1976.
2. A u l d , D. S. and H o l m q u is t , B.: Carboxypeptidase A. Differences in the mechanisms of
ester and peptide hydrolysis. Biochemistry
13:4355-4361, 1974.
3. B osron , W. F., Ke n n e d y , F. S., and V a l l e e ,
B. L.: Zinc and magnesium content of alkaline
phosphatase from Escherichia coli. Biochemis­
try 24:2275-2282, 1975.
4. B r a d s h a w , R. A., E r i c c s o n , L. H., W a l s h , K.
A., and N e u r a t h , H.: The amino acid se­
quence of bovine carboxypeptidase A. Proc.
Nat. Acad. Sei. 63:1389-1394, 1969.
5. B r ä n d e n , C. I., J Ö 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.
The Enzymes, vol. XI. Boyer, P. D., ed. New
York, Academic Press, pp. 103-190, 1975.
6. D ix o n , N. E., G a z z o l a , C., Bl a k e l e y , R. L.,
and ZERNER, B.: Jack bean urease (EC3.5.1.0).
A metalloenzyme. A simple biological role for
nickel? J. Amer. Chem. Soc. 97:4131-4133,
1975.
7. D r u m , D . E. and V a l l e e , B. L.: Differential
chemical reactivities of zinc in horse liver al­
cohol dehydrogenase. Biochemistry 9:40784086, 1970.
8. F al c h u k , K. F., F a w c et t , D. W., and V al ­
l e e , B. L.: Role of zinc in cell division of
Euglena gracilis. J. Cell. Sei. 17:57-78, 1975.
9. F a l c h u k , K. F ., K r i s h a n , A., and V a l l e e , B.
L.: DNA distribution in the cell cycle of Eu­
glena gracilis. Cytofluorometry of zinc defi­
cient cells. Biochemistry 24:3439-3444, 1975.
ROLE OF METALS IN ENZYME ACTIVITY
10. F l o h e , L., G u n z l e r , W. A., an d Sc h o c k , N. H.:
G lu ta th io n e peroxidase: A s e le n o e n zy m e . F ed.
E u ro p e a n B io c h e m . Soc. L etters 32:132-136,
1973.
11. J a c o b s o n , G. B. and S t a r k , G. B.: Aspartate
transcarbamylases. The Enzymes, vol. IX.
Boyer, P. D., ed. New York, Academic Press,
pp. 225-308, 1973.
12. J o h a n s e n , J. T. and V a l l e e , B. L.: Differ­
ences betw een the conform ation of
arsanilazotyrosine 248 of carboxypeptidase in
the crystalline state and in solution. Proc. Nat.
Acad. Sci. 68:2532-2535, 1971.
13. J o h a n s e n , J. T. and V a l l e e , B. L.: Conforma­
tions of arsanilazotyrosine 248 carboxypepti­
dase Aa,p,y. Comparison of crystals and so­
lutions. Proc. Nat. Acad. Sci. 70:2006-2010,
1973.
14. LEHNINGER, A. L.: Bole of metal ions in en­
zyme systems. Physiol. Bev. 30:393-429, 1950.
15. Li, T. K. and V a l l e e , B. L.: Trace elements,
Section B. The biochemical and nutritional
role of trace elements. Modem Nutrition in
Health and Disease. Goodhart, R. S. and Shils,
M. E., eds. Philadelphia, Lea and Febiger, pp.
372-399, 1973.
16. L i p s c o m b , W . N ., H a r t s u c k , J. A., R e e k e ,
G . N., J r ., Q u io c h o , F. A., B e t h g e , P. H ., L u d ­
w i g , M . L ., S t e i t z , T. A., M u i r h e a d , H .,
and C o p p o l a , J. C .: The structure of carboxy­
peptidase A. V II. The 2.0 A resolution studies
of the enzyme and of its complex with glycyl
tyrosine, and m echanistic deductions.
Brookhaven Symp. Biol. 21:24-90, 1968.
17. M a h l e r , H. R.: Interrelationships with en­
zymes. Mineral Metabolism, vol. 1, Part B.
Comas, C. L. and Bronner, F., eds., New York,
Academic Press, pp. 743-879, 1961.
18. M OORE, C. V.: Major Minerals. Section C, Iron.
Modern Nutrition in H ealth and Disease.
Goodhart, R. S. and Shils, M . E., eds. Philadel­
phia, Lea and Febiger, pp. 297-323, 1973.
19. N o r t h r o p , D. B. and W o o d , H. G.: Transcar­
boxylase V. The presence of bound zinc and
cobalt. J. Biol. Chem. 244:5801-5807, 1969.
129
20. O ’D e l l , B. L.: Biochemistry and physiology of
copper in vertebrates. Trace Elem ents in
Human Health and Disease, Zinc and Copper.
Prasad, A. S., ed., New York, Academic Press, pp.
391-413, 1976.
21. R lO R D A N , J. F.: Biochemistry of zinc. The Med­
ical Clinics of North America. 60:661-674,1976.
22. B i o r d a n , J. F. and V a l l e e , B. L.: The func­
tional role o f metals in m etalloenzym es.
Protein-Metal Interactions. Friedman, M., ed.
Advances in Exp. Med. Biol. 48:33-58, 1974.
23. R i o r d a n , J. F. and VALLEE, B. L.: Structure
and function of zinc metalloenzymes. Trace
Elements in Human Health and Disease, Vol.
1, Zinc and Copper. Prasad, A. S., ed., New
York, Academic Press, pp. 227-256, 1976.
24. SCRUTTON, M. C.: Metal enzymes. Inorganic
Biochemistry, vol. 1. Eichorn, G. L., ed.
Amsterdam, Elsevier, pp. 381-437, 1973.
25. S im p s o n , R. T. and V a l l e e , B. L.: Two differ­
entiable classes of metal atoms in alkaline
phosphatase of Escherichia coli. Biochemistry
7:4343-4350, 1968.
26. S t e p h e n s , R. S., J e n t o f t , J. E., and B r y a n t ,
B. G.: 35C1 nuclear magnetic resonance inves­
tigation of carboxypeptidase A. J. Amer. Chem.
Soc. 96:8041-8045, 1974.
27. K n a p p e i s , G. G. and C A R L S O N , F.: Ultrastruc­
ture of Z-disc in skeletal muscle. J. Cell. Biol.
23:323-335, 1962.
28. V a l l e e , B. L.: Zinc and metalloenzymes. Adv.
Protein Chem. 10:317-334, 1955.
29. V A L L E E , B. L.: C obalt substituted zinc
metalloenzymes. Metal Ions in Biological Sys­
tems. Dhar, S. K., ed. New York, Plenum, pp.
1-12, 1973.
30. V a l l e e , B. L.: Zinc biochemistry: a perspec­
tive. Trends in Biochem. Sci. 2:88-91, 1976.
31. V a l l e e , B. L. and N e u r a t h , H.: Carboxypep­
tidase: a zinc metalloprotein. J. Amer. Chem.
Soc. 76:5006-5007, 1954.
32. V a l l e e , B. L. and W i l l i a m s , R. J. P.: Enzyme
action: views derived from metalloenzyme
studies. Chem. in Britain 4:397-402, 1968.