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ABSTRACT Zinc is required for the activity of ⬎ 300 enzymes, covering all six classes of enzymes. Zinc binding
sites in proteins are often distorted tetrahedral or trigonal bipyramidal geometry, made up of the sulfur of cysteine,
the nitrogen of histidine or the oxygen of aspartate and glutamate, or a combination. Zinc in proteins can either
participate directly in chemical catalysis or be important for maintaining protein structure and stability. In all
catalytic sites, the zinc ion functions as a Lewis acid. Researchers in our laboratory are dissecting the determinants
of molecular recognition and catalysis in the zinc-binding site of carbonic anhydrase. These studies demonstrate
that the chemical nature of the direct ligands and the structure of the surrounding hydrogen bond network are
crucial for both the activity of carbonic anhydrase and the metal ion affinity of the zinc-binding site. An under-
standing of naturally occurring zinc-binding sites will aid in creating de novo zinc-binding proteins and in designing
new metal sites in existing proteins for novel purposes such as to serve as metal ion biosensors. J. Nutr. 130:
Zinc was first shown to be required for the growth of the Properties of zinc
mold Aspergillus niger by Raulin in 1869. Since then, zinc has
been demonstrated to be essential for the growth, develop- The inherent chemical potential and reactivity of zinc are
ment and differentiation of all types of life, including micro- not exceptional compared with those of other metals (Cotton
organisms, plants and animals (Vallee 1986). After iron, zinc and Wilkinson 1988). However, unlike other first-row transi-
is the second most abundant trace metal in the human body; tion metals (e.g., Sc2⫹, Ti2⫹, V2⫹, Cr2⫹, Mn2⫹, Fe2⫹, Co2⫹,
an average 70-kg adult human contains 2.3 g of zinc (Mc- Ni2⫹ and Cu2⫹), the zinc ion (Zn2⫹) contains a filled d orbital
Cance and Widdowson 1942). The first zinc metalloenzyme, (d10) and therefore does not participate in redox reactions but
carbonic anhydrase II (CA3 II, EC 4.2.1.1), was discovered in rather functions as a Lewis acid to accept a pair of electrons
1940 by Keilin and Mann. Since then, ⬎ 300 zinc enzymes (Williams 1987). This lack of redox activity makes Zn2⫹ a
covering all six classes of enzymes and in different species of all stable ion in a biological medium whose potential is in con-
phyla have been discovered (Christianson 1991, Coleman stant flux. Therefore, the zinc ion is an ideal metal cofactor for
1992, Vallee and Auld 1990a). In most cases, the zinc ion is an reactions that require a redox-stable ion to function as a Lewis
essential cofactor for the observed biological function of these acid–type catalyst (Butler 1998), such as proteolysis and the
metalloenzymes. Furthermore, the biological functions of zinc, hydration of carbon dioxide. Furthermore, due to the filled
which are versatile and observed in many tissues, are most d-shell orbitals, Zn2⫹ has a ligand-field stabilization energy of
often associated with proteins. zero (Huheey et al. 1993) in all liganding geometries, and
hence no geometry is inherently more stable than another.
This lack of an energetic barrier to a multiplicity of equally
1
Presented at the international workshop “Zinc and Health: Current Status
accessible coordination geometries can be used by zinc met-
and Future Directions,” held at the National Institutes of Health in Bethesda, MD, alloenzymes to alter the reactivity of the metal ion and may be
on November 4 –5, 1998. This workshop was organized by the Office of Dietary an important factor in the ability of Zn2⫹ to catalyze chemical
Supplements, NIH and cosponsored with the American Dietetic Association, the
American Society for Clinical Nutrition, the Centers for Disease Control and
transformations accompanied by changes in the metal coordi-
Prevention, Department of Defense, Food and Drug Administration/Center for nation geometry. Nevertheless, in all zinc metalloenzymes
Food Safety and Applied Nutrition and seven Institutes, Centers and Offices of the studied to date, the binding geometry observed most often is a
NIH (Fogarty International Center, National Institute on Aging, National Institute of slightly distorted tetrahedral (Scheme 1) with the metal ion
Dental and Craniofacial Research, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institute on Drug Abuse, National Institute of coordinating three or four protein side chains. However, five-
General Medical Science and the Office of Research on Women’s Health). Pub- coordinate distorted trigonal bipyramidal geometry has been
lished as a supplement to The Journal of Nutrition. Guest editors for this publi- observed in the metal sites of Zn-substituted astacin (EC
cation were Michael Hambidge, University of Colorado Health Science Center,
Denver; Robert Cousins, University of Florida, Gainesville; Rebecca Costello, 3.4.24.21) (Bode et al. 1992), two CA II (EC 4.2.1.1) mutants
Office of Dietary Supplements, NIH, Bethesda, MD; and session chair, Craig (H119D and H94D) (Ippolito and Christianson 1994, Kiefer et
McClain, University of Kentucky, Lexington. al. 1993a), the bimetal sites of purple acid phosphatase (EC
2
To whom correspondence should be addressed at Chemistry Department,
University of Michigan, 930 N. University, Ann Arbor, MI 48109. 4.2.1.1) (Klabunde et al. 1996) and the trimetal sites of phos-
3
Abbbreviation used: CA, carbonic anhydrase. pholipase C (EC 3.1.4.3) (Hough et al. 1989). In addition,
1437S
1438S SUPPLEMENT
TABLE 1
Comparison of the zinc ligands1 (L1, L2, L3 and L4) and the spacers (X, Y and Z)
between zinc ligands in catalytic and structural zinc sites
L1 X L2 Y L3 Z L4 Solvent
Catalytic zinc
Oxidoreductases2
Alcohol dehydrogenase (horse liver)3 C37 20 H59 106 C174 H2O
Alcohol dehydrogenase (Thermoanaerobium
brockii)4 C37 21 H59 90 D150 NA5
Hydrolases
Carboxypeptidase A (bovine)6,7 H69 2 E72 123 H196 H2O
Thermolysin (Bacillus thermoproteolyticus)8,9 H142 3 H146 19 E166 H2O
DD carboxypeptidase (Streptomyces albus)10 H196 2 H193 40 H152 H2O
Astacin (crayfish)11 H92 3 H96 5 H102 H2O
-Lactamase (Bacillus cereus)12 H86 1 H88 121 H210 H2O
Cytidine deaminase (Escherichia coli)13 C132 2 C129 26 H102 H2O
Alkaline phosphatase (E. coli)14 D327 3 H331 80 H412 H2O
Adenosine deaminase (murine)15 H15 1 H17 196 H214 80 D295 H2O
Lyases
Carbonic anhydrase II (human)17,16 H94 1 H96 22 H119 H2O
Carbonic anhydrase (spinach)18 C213 2 H210 59 C150 H2O
1 Included in this table are representative enzymes in which the active site ligands differ. The protein ligands are shown using the one-letter amino
acid codes of: H, histidine; C, cysteine; D, aspartate; and E, glutamate.
2 The relatively long spacer between L1 and L2 is unusual, and it may be due to the requirements of NAD(H) cofactor binding at the active site.
5 Not available. The zinc ligands were determined by mutations that abolish both the zinc-binding affinity and the catalytic activity.
19 The zinc site of carbonic anhydrase of M. thermophila is composed of three histidines: His81 and His122 from one subunit and His117 from the
neighboring subunit.
21 The type of zinc ligands of cobalamin-independent methionine synthase were determined by EXAFS to be a combination of two S atoms and
two N/O atoms.
References: 3 Cedergren-Zeppezauer et al. 1985, 4 Bogin et al. 1997, 6 Rees et al. 1983, 7 Vallee and Auld 1990a, 8 Matthews et al. 1972, 9 Pauptit
et al. 1988, 10 Dideberg et al. 1982, 11 Bode et al. 1992, 12 Sutton et al. 1987, 13 Betts et al. 1994, 14 Kim and Wyckoff 1991, 15 Wilson et al. 1991,
16 Hewett-Emmett and Tashian 1996, 17 Liljas et al. 1972, 18 Bracey et al. 1994, 19 Kisker et al. 1996, 20 Park et al. 1997, 21 Peariso et al. 1998,
22 Myers et al. 1993b, 23 Honzatko et al. 1982, 24 Pavletich and Pabo 1991, 25 Luisi et al. 1991, 26 Fuiji et al. 1997 and 27 Zhou et al. 1999.
site and lower the entropic cost of binding the metal ion repair protein with an unusual zinc site (Myers et al. 1992).
(Christianson 1991). These interactions between metal ion The tetrahedral Cys4 zinc polyhedron in the N-terminal do-
and ligand have been proposed to orient the metal ligands, main of Ada is important to stabilize the tertiary fold. How-
enhance the electrostatic interaction between metal and li- ever, in addition, this zinc site catalyzes the direct, irreversible
gand and modulate the zinc-water pKa (Argos et al. 1978, transfer of a methyl group from the Sp diastereomer of DNA
Christianson 1991). methylphosphotriester to the sulfur of Cys69 in the coordina-
Interestingly, the catalytic metal in two zinc metallopro- tion sphere (Myers et al. 1993a). This conversion of the
teins is believed to have a central role in regulation of enzyme thiolate metal ligand to a more weakly bound thioether ligand
activity. The first example is stromelysin (EC 3.4.24.17), on methyl transfer has been proposed to propel structural
which is produced as an inactive proenzyme and activated by changes that reveal the sequence-specific DNA binding con-
proteolysis of ⬇ 80 amino acids from the N terminus (Van formation of the protein (Myers et al. 1993a). In this confor-
Wart and Birkedal-Hansen 1990). This activation involves mation, Ada functions as a transcription factor, inducing genes
the replacement of a thiolate zinc ligand with a water mole- that confer resistance to methylating agents. The proposed
cule, yielding the catalytically active His3H2O zinc site role of the zinc ion in Ada, coordinating the cysteine thiolate
(Gooley et al. 1993, Holz et al. 1992, Salowe et al. 1992, Van to lower the pKa and enhance the reactivity of this group
Wart and Birkedal-Hansen 1990). (Matthews and Goulding 1997, Myers et al. 1993), is similar to
The second case is the Escherichia coli Ada protein, a DNA the role being proposed for zinc in several enzymes that cata-
1440S SUPPLEMENT
TABLE 2
Features of catalytic zinc and structural zinc status
Cocatalytic Zn5
FIGURE 1 The zinc-binding site of CA II. The first two ligands, H94
and H96, are on the same stand of the -sheet. The third ligand, H119,
SCHEME 2 is on the neighboring strand of the -sheet.
FUNCTION AND MECHANISM OF ZINC METALLOENZYMES 1441S
the zinc site have not yet been completely defined for any zinc
metalloenzymes, although CA has been studied in the greatest
detail (Christianson and Fierke 1996). The de novo design of
zinc sites using solely the geometry of structurally characterized
sites (Hellinga 1998, Hellinga et al. 1991, Regan 1995) has
yielded metal sites with the correct geometry but decreased
metal affinity and little or no catalytic activity, demonstrating
our ignorance about the role of the protein in modulating the
reactivity of the bound metal.
Example of catalytic zinc site. CA II. CA is a ubiquitous
zinc metalloenzyme that catalyzes the reversible hydration of
carbon dioxide. In mammals, more than seven isozymes have
been identified, and the isozyme CA II has the highest specific
activity (Silverman and Lindskog 1988, Silverman and Vin-
cent 1984). CA II plays a variety of physiological roles, in-
cluding promoting CO2 exchange in the erythrocytes, kidney
and lung; contributing to acid-base homeostasis; and promot-
ing HCO3⫺ secretion (Sly and Hu 1995).
The basic catalytic mechanism of CA was established from
studies of bovine CA and human CAs I and II (Silverman and
Lindskog 1988, Silverman and Vincent 1984). Although ad-
SCHEME 4
active form of the enzyme is a trimer of left-handed -helices, variants reveal that either the zinc-binding geometry is
and the zinc-containing active sites are located at the inter- changed from tetrahedral to trigonal bipyramidal (H94N CA II
faces between two monomers, yet the active site itself is and H119N CA II) or the position of the zinc-bound water is
strikingly similar to that of CA II. The zinc-binding site is moved (H119Q CA II) (Lesburg et al. 1997). The bulky
made up of His81 and His122 from one monomer and His117 histidine ligands (especially H119) may play a role in disfavor-
from the neighboring monomer along with a coordinated ing higher coordination numbers, and therefore stabilizing a
water. Similarly, the shell of “indirect ligands” is also found; low coordination number, for the active site zinc ion of native
the hydrogen bond acceptors of CA II, Q92, N244 and E117, CA II. This decreased coordination number should both de-
have their analogs in the CA of M. thermophila, D61, G123 and press the pKa of zinc-bound solvent and increase its reactivity
D76, respectively. In the -CA family, no high resolution (Bertini et al. 1990). Furthermore, the metal affinity of the
structure is currently available. However, because sulfon- variants with carboxamide ligands is significantly compro-
amides inhibit plant-type CAs (Pocker and Ng 1974) as they mised (Lesburg et al. 1997). Taken together, these data indi-
do in mammalian CAs, it is presumed that the zinc bound to cate that the neutral histidine ligands of the zinc-binding site
the plant enzyme is also catalytic. Furthermore, the zinc poly- optimize the electrostatic environment of the active site to
hedron of spinach CA, identified by EXAFS (extended X-ray maintain high catalytic activity and high zinc affinity in CA
absorption fine structure) and mutagenesis studies, reveals a II.
tetracoordinate His1Cys2Wat1 zinc site (Bracey et al. 1994, The indirect ligands. Structure-based dissection of the
Rowlett 1984), supporting the suggestion that the zinc is indirect ligands of CA II (Huang et al. 1996, Kiefer et al. 1995,
catalytic. Lesburg and Christianson 1995) implicated these residues in
Investigation of CA II. The determinants of metal affinity “fine tuning” the electrostatic environment of the zinc ion and
and catalysis in the zinc-binding site of CA have been inves- enhancing zinc-binding affinity. The zinc affinity of variants
The hydrophobic core. The role of highly conserved aro- Cedergren-Zeppezauer, E. S., Andersson, I., Ottonello, S. & Bignetti, E. (1985)
X-ray analysis of structural changes induced by reduced nicotinamide ade-
matic residues near the zinc-binding site of CA II in affecting nine dinucleotide when bound to cysteine-46-carboxymethylated liver alcohol
metal ion binding has also been examined. Residues F93, F95 dehydrogenase. Biochemistry 24: 4000 – 4010.
and W97 are located along the -strand containing the direct Chakrabarti, P. (1989) Geometry of interaction of metal ions with sulfur-
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and slow zinc dissociation rate of CA II (Hunt and Fierke residues in protein structures. Prot. Eng. 4: 57– 63.
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at these positions result in up to eightfold reductions in Zn/Co amide groups in protein structures. Prot. Eng. 4: 49 –56.
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