Download the interaction of metal ions with enzymes

Name :Etso Erhuvwu Faith
Matric number :13/sci03/006
Dept: Biochemistry
Level:400
BCH 413
Assignment:
The role of metalloenzymes in biological systems
A metalloenzyme is simple any enzyme containing tightly bound metal atoms.Enzymes
affected by metal ions have been operationally defined as either metalloenzymes or metalenzyme complexes(Vallee,1973). A metalloenzyme contains a firmly bound, stoichiometric
quantity of a metal as an integral part of the protein molecule. Removal of the metal by
treatment 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 isolated, and the enzyme may exhibit partial activity in the
absence of the metal ion. Obviously, the difference between these two classes of metalenzyme systems depends on the magnitude of the metalprotein stability constant which can
be a function of the metal atom as well as environmental conditions such as pH, buffer and
ionic strength. The metalloenzymes are better suited for elucidation of the metal protein
interaction and for extrapolating such information to the understanding 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 understanding of catalytic phenomena and general mechanisms
of catalysis by metalloenzymes.
Certain metals have long been recognized to have important biological functions primarily as
a consequence of nutritional investigations (LEHNINGER,1950).. 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 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
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, molybdenum, nickel, selenium and zinc (table I).
Aside from its role in vitamin B12, cobalt, to date, has been found to be component of but
one enzyme, the biotin-dependent, zinc-containing oxaloacetate transcarboxylase of
Propionibacterium shermanii. 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.(Moore,1973)Manganese has been identified as a
component of pyruvate carboxylase from chicken liver and is present in Escherichia coli
superoxide dismutase.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. 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.(Flohe et
al.1973 )
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. 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.
THE INTERACTION OF METAL IONS WITH ENZYMES
A number of schemes have been presented to describe the types of interactions that can occur
between metals, enzyme 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 typically observed with metal-activated enzymes.
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 terminalpeptide bond, i.e., the one that is
susceptible to hydrolysis. However, even though some kind of metal-substrate bond may be formed,
the metal does not appear to be essential for peptide substrate 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 differences that have
been observed for carboxypeptidase acting on ester and peptide substrates, respectively.(Auld and
Holmquist,1974)
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 enzymes where the
metal-protein interaction is more readily controlled by manipulation of the ambient metal ion
concentration. It should be emphasized that these schemes are not all mutually exclusive and that
some metalloenzymes are known to contain functionally different classes of metal ions.
Figure 1. Interactions between metal (M), enzyme
(E) and substrate (S).
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 participates directly in the electron
transfer process and undergoes a cyclic change in oxidation state. Often times the free metal is
capable of catalysis by itself as with the iron-promoted decomposition of hydrogen peroxide although
in this case catalase is at least a million times more effective than iron alone. Thus, the protein
component of a metalloenzyme contributes many of the critical aspects of the catalytic mechanism.
Zinc, on the other hand, does not undergo a change in oxidation state during enzymatic catalysis even
though it participates in oxidoreduction reactions, e.g., as a component of alcohol dehydrogenase. The
zinc cation has a stable, d10 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 chemical bonds and thus promote the transfer of atoms or groups. Substitution
reactions of simple metal chelates generally proceed via intermediates with an open coordination
position or a distorted coordination sphere. Zinc (and also cobalt) can readily accept a distorted
geometry and, hence, would appear to be well suited to participate in substitution reactions as, for
instance, in carbonic anhydrase, carboxypeptidase and alkaline phosphatase.
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(Jacobson and Stark,1973). 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 s tabilize 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.
REFERENCES
Auld , D. S. and Holmquist, B. Carboxypeptidase A. Differences in the mechanisms of ester and
peptide hydrolysis. Biochemistry 13:4355-4361, 1974.
Jacobson , G. B. and Stark , G. B.: Aspartate transcarbamylases. The Enzymes, vol. IX. Boyer, P. D., ed.
New York, Academic Press, pp. 225-308, 1973.
LEHNINGER, A. L. Role of metal ions in enzyme systems. Physiol. Bev. 30:393-429, 1950.
MOORE, C. V. Major Minerals. Section C, Iron. Modern Nutrition in Health and Disease. Goodhart, R.
S. and Shils, M . E., eds. Philadelphia, Lea and Febiger, pp. 297-323, 1973
VALLEE, B. L.: Cobalt substituted zinc metalloenzymes. Metal Ions in Biological Systems. Dhar, S. K.,
ed. New York, Plenum, pp. 1-12, 1973.