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Zinc and Health: Current Status and Future Directions

Function and Mechanism of Zinc Metalloenzymes1


Keith A. McCall, Chih-chin Huang and Carol A. Fierke2
Duke University Medical Center, Durham, NC 27710

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:

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1437S—1446S, 2000.

KEY WORDS: ● zinc ● carbonic ● anhydrase ● metalloenzyme ● zinc-binding sites

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,

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.

1437S
1438S SUPPLEMENT

zinc-binding polyhedron contains at least one water molecule


in addition to three or four protein ligands (Vallee and Auld
1992a, 1992b). This feature is diagnostic of a catalytic zinc site
compared with a structural zinc site, where the metal polyhe-
dron is saturated with protein side chains and this difference
can be detected by a number of spectroscopic and structural
techniques. The zinc-bound water is a critical component for
a catalytic zinc site, because it can be either ionized to zinc-
bound hydroxide (as in CA), polarized by a general base (as in
SCHEME 1 carboxypeptidase A) to generate a nucleophile for catalysis or
displaced by the substrate (as in alkaline phosphatase; EC
3.1.3.1) (Scheme 2) (Vallee and Auld 1990a and 1993b). In
five-coordinate geometry has been suggested for the reaction the zinc hydrolases and lyases, such as the zinc proteases and
intermediate in CA (Christianson and Fierke 1996, Lindskog CAs, the zinc ion serves as a powerful electrophilic catalyst by
1997) and carboxypeptidase A (EC 3.4.17.1) (Christianson providing all or a combination of the following: (1) an acti-
and Lipscomb 1989). A final important property of Zn2⫹ that vated water molecule for nucleophilic attack, (2) polarization
makes it well suited as a catalytic cofactor is that ligand of the carbonyl of the scissile bond and (3) stabilization of the
exchange is rapid (Cotton and Wilkinson 1988), allowing for negative charge in the transition state (Christianson and Cox,
the rapid product dissociation required for efficient turnover. 1999, Lovejoy et al. 1994, Silverman and Lindskog 1988,
Early examinations of the coordination preferences of zinc Vallee and Galdes 1984).
ions concluded that zinc preferentially bound to sulfur ligands The X-ray structures of catalytic zinc enzymes from four of

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due to the predominance of zinc sulfides in zinc ores, e.g., the six classes of enzymes (oxidoreductases, transferases, hy-
wurtzite, a hexagonal ZnS array containing zinc in a distorted drolases and lyases) have been determined, and they define the
tetrahedral coordination geometry, and zinc blende, a cubic features of catalytic zinc-binding sites. Unlike the structural
ZnS array containing zinc in a perfect tetrahedral coordination sites, the metal ion in catalytic sites is generally coordinated to
geometry (Cotton and Wilkinson 1988, Vallee and Auld the side chain of three amino acid residues, a combination of
1990a). However, after an examination of a variety of small histidine, glutamate, aspartate and cysteine, and a solvent
molecule coordination complexes in solution, Pearson (1963) molecule completes the tetrahedral coordination sphere (Ta-
classified zinc as a “borderline” metal, meaning that Zn2⫹ does ble 1) (for reviews, see Christianson 1991, Jernigan et al. 1994,
not consistently act either “hard” (not very polarizable) or Vallee and Auld 1990b). However, the zinc polyhedra of
“soft” (highly polarizable) and does not have a strong prefer- adenosine deaminase (EC 3.5.4.4) (Wilson and Quiocho
ence for coordinating with either oxygen, nitrogen or sulfur 1993, Wilson et al. 1991) and astacin (EC 3.4.24.21) (Gomis-
atoms. In protein zinc-binding sites, the zinc ion is coordinated ruth et al. 1993) are composed of four amino acid side chains
by different combinations of protein side chains, including the and a solvent molecule with a trigonal bipyramidal geometry.
nitrogen of histidine, the oxygen of aspartate or glutamate and In catalytic zinc sites, histidine is the most frequently ob-
the sulfur of cysteine; among these, histidine is most com- served ligand, distantly followed by glutamic acid and then
monly observed, followed by cysteine (Gregory et al. 1993) aspartic acid and cysteine. In non– coenzyme-dependent zinc
(Table 1). Other, much more rarely observed ligands include enzymes, a short spacer with a rigid arrangement of one to
the hydroxyl of tyrosine, the carbonyl oxygen of the protein three amino acids intervening between the first two ligands, L1
backbone and the carbonyl oxygen of either asparagine or and L2 (Table 1), may constitute a nucleus for the zinc-
glutamine. The varied ligands and coordination geometries in binding site (i.e., CA site, Fig. 1). The third ligand, L3, is
zinc metalloenzymes result in zinc-binding sites with a broad separated from L2 by a longer spacer whose length varies
range of stability constants, reactivities and functions. greatly (5–200 amino acids) and may be responsible for the
In zinc proteins, the major role of the zinc ion can be spatial formation of the active site, allowing some flexibility to
catalytic, cocatalytic or structural. In a catalytic zinc site, the the coordination sphere.
zinc ion directly participates in the bond-making or -breaking The majority of histidine zinc ligands found in zinc protein
step. In a cocatalytic zinc site, there are several metal ions structures coordinate zinc through the N⑀ atom (Chakrabarti
bound in proximity to one another, where one plays a catalytic 1990a, Glusker 1991), although coordination with N␦ atoms
role and the other metal ions enhance the catalytic activity of has also been observed. For these interactions, the metal ion
the site (Vallee and Auld 1993a). Finally, in structural zinc prefers a head-on and in-plane approach to the sp2 lone pair of
sites, the zinc ion mainly stabilizes the tertiary structure of the the nitrogen atom (Vedani and Huhta 1990) (Scheme 3).
enzyme in a manner analogous to disulfide bonds. In all cases, Carboxylate-zinc interactions with syn-stereochemistry are ob-
removal of the bound zinc can lead to a loss of enzymatic served more frequently than those with anti-stereochemistry,
activity. A systematic analysis of the structure and function of and the zinc ion displays a preference to be in the plane of the
a number of zinc proteins has established distinct features of carboxyl (Carrell et al. 1988, Chakrabarti 1990). A stereo-
catalytic and structural zinc sites, as described later (Table 2) chemical analysis of cysteine-zinc interactions in the
(Arnold and Haymore 1991, Coleman 1992, Vallee and Auld Brookhaven Data Bank revealed that the average sulfur-zinc
1993a, 1990b). As understanding of the biochemical role of distance is 2.1 Å, the average C␤-S-zinc angle is 112 degrees
zinc in these biological macromolecules increases, the connec- and the C␣-C␤-S-zinc torsion angle distribution is trimodal
tion between the detailed biochemical functions and physio- with peaks at ⫾ 90 and 180 degrees (Chakrabarti 1989).
logical phenotypes can be established. A feature common to all zinc sites is that the metal ion is
Catalytic zinc sites. A catalytic zinc ion is located at the surrounded by a shell of hydrophilic groups that is embedded
active site of an enzyme, where it participates directly in the within a larger shell of hydrophobic groups (Yamashita et al.
catalytic mechanism, interacting with the substrate molecules 1990). In addition, the amino acid side chains serving as zinc
undergoing reaction. A unique feature for a catalytic zinc site ligands in these structures often make hydrogen bond contacts
is the existence of an open coordination sphere; that is, the with other residues, perhaps to preorder the metal ion binding
FUNCTION AND MECHANISM OF ZINC METALLOENZYMES 1439S

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

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Carbonic anhydrase (Methanosarcina
thermophila)19 H81 35 H117 NA19 H122 H2O
Novel catalytic zinc sites
Transferases
Protein farnesyltransferase (rat)20 D297 1 C299 62 H362 H2O
Cobalamin-dependent methionine synthase
(E. coli)21 C247 62 C310 C311 N/O atom
Cobalamin-independent methionine synthase
(E. coli)21,27 H641 1 C643 82 C726 N/O atom
Nonenzymatic
Ada repair protein (E. coli)22 C38 3 C42 26 C69 2 C72 No
Structural zinc
Alcohol dehydrogenase (horse liver)3 C97 2 C100 2 C103 7 C111 No
Aspartate carbamoyltransferase (E. coli)23 C109 4 C114 23 C138 2 C141 No
Zinc finger (Zif268) (mouse)24 C7 5 C12 12 H25 4 H29 No
Glucocorticoid receptor (rat)25 C440 2 C443 8 C457 2 C460 No
Ferrodoxin (Sulfolobus sp.)26 H16 2 H19 14 H34 41 D76 No

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

Catalytic Zn4 Catalytic Zn Noncatalytic Zn Structural Zn4

Coordination sphere Open Open Closed or bridging water Closed


Ligands1 H, D, E, C, wat H, D, E, wat H, D, S, N/O atoms, wat C, H, (D)3
Ligands (n) 4, 5 4 5 4
Binding geometry Asymmetrical: distorted Distorted tetrahedral or Slightly distorted Symmetrical: tetrahedral
tetrahedral or distorted octahedral minus trigonal bipyramidal2
trigonal bipyramidal one geometry
Location of ligands Rigid: ␣-helix or ␤-sheet Nonrigid: loop, ␣-helix or ␤-sheet Nonrigid: loop, ␣-helix
or ␤-sheet
Spacing between Regular Regular Irregular Irregular
ligands

1 Most common ligands are in bold.


2 Mg is octahedral.
3 D observed in only one case: ferrodoxin from Sulfolobus sp.
4 Vallee and Auld 1990b.
5 Vallee and Auld 1993a.

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lyze S-alkylation reactions, such as cobalamine-dependent me- sites (Table 1). However, the second zinc (Zn2) and the third
thionine synthase (EC 2.1.1.13), cobalamine-independent metal (Zn3/Mg) ion sites may have unusual ligands such as the
methionine synthase (EC 2.1.1.14), methanol:coenzyme M oxygen of serine or threonine or the nitrogen of the N-
methyltransferase (EC 2.1.1.86) and protein farnesyltrans- terminal amino group. An additional distinctive feature is the
ferase (EC 2.5.1.21) (Gonzalez et al. 1996, Goulding and existence of one or more bridging ligands (either aspartate or
Matthews 1997, Hightower et al. 1998, Huang et al. 1997, water or both) between the second zinc ion (Zn2) and the
LeClerc and Grahame 1996). This class of zinc metallopro- third zinc ion (Zn3) or magnesium ion (Zn3/Mg), which often
teins may indicate a new catalytic function of the zinc ion: to leads to pentacoordinate geometry. The distance between the
enhance the nucleophilicity of a thiol group at neutral pH catalytic zinc ion (Zn1) and the second zinc ion (Zn2), at 4 –5
(Hightower and Fierke 1999, Matthews and Goulding 1997). Å, is shorter than that between the catalytic zinc ion and the
Cocatalytic zinc sites. In multimetal enzymes, the two or third zinc ion (Zn3), at 6 –7 Å (Fig. 2). For phosphate ester
more zinc (or other metal) atoms may operate in concert to hydrolyzing enzymes, the product phosphate interacts with all
enhance catalysis. A class of catalytic zinc sites, called cocata- three metals, displacing the weak unusual ligands in the sec-
lytic zinc sites, has been defined in which two or more zinc ond and third zinc sites to facilitate catalysis (Vallee and Auld
atoms are in close proximity to one another (Vallee and Auld 1993b). These sites are termed “cocatalytic” because all three
1992a, 1992b, 1993b). This group of enzymes includes alkaline metals play crucial roles in catalysis despite only the zinc
phosphatase (with two zinc ions and one magnesium ion), activating the attacking water being termed “catalytic.”
phospholipase C (three zinc ions), nuclease P1 (EC 3.1.30.1; Structural zinc sites. In structural zinc sites, the metal ion
three zinc ions) and leucine aminopeptidase (two zinc ions) is coordinated by four amino acid side chains, usually in a
(Vallee and Auld 1993b). A representative structure (phos- tetrahedral geometry, so that solvent is excluded as an inner
pholipase C) is shown in Figure 2. The first zinc ion, desig- sphere ligand (for reviews, see Vallee and Auld 1990b, Vallee
nated as the catalytic zinc (Zn1 in Fig. 2), contains a bound et al. 1991). Cysteine is by far the ligand observed most
water that is essential for catalysis and has an His2Glu metal frequently in these sites, with histidine also being present in
polyhedron similar to those found in other single catalytic zinc many cases and aspartate being present in one case (Lovejoy et

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-

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ditional CA isozymes and families have been discovered in
recent years, the main features of the catalytic mechanism of
the mammalian enzyme are retained (Lindskog 1997). The
mechanism of CO2 hydration, catalyzed by CA II, can be
separated into two steps (Scheme 4). In the first step, zinc-
bound hydroxide attacks the carbonyl carbon of CO2 to form
zinc-bound bicarbonate; bicarbonate is subsequently displaced
with water by a ligand-exchange step. In the second step, H⫹
SCHEME 3
is transferred from zinc-bound water to external buffer via a
shuttle group (H64 in CA II) to regenerate the catalytically
active species, the zinc-bound hydroxide (Silverman and Lind-
al. 1994, Spurlino et al. 1994). In contrast to catalytic zinc skog 1988).
sites, these sites contain no regular pattern of spacer length The high resolution x-ray crystal structure of human CA II
between the protein zinc ligands, and the ligands can be was first solved in 1972 (Liljas et al. 1972) and further refined
located on a flexible loop rather than in a rigid secondary to 2 Å (Eriksson et al. 1988) and then to 1.54 Å (Hakansson
structure. The high stability constants of these tetradentate et al. 1992) (Fig. 3). The active site cavity is ⬇15 Å wide at
zinc complexes ensure both local and overall structural stabil- the entrance and ⬇15 Å in depth. The zinc ion is coordinated
ity similar to that provided by disulfides (Vallee and Auld to three histidine residues, N⑀ of H94, N␦ of H96 and N⑀ of
1990b). This enables proteins containing structural zinc atoms H119, and a water molecule in a tetrahedral manner at the
to perform a wide range of functions.
Importance of further study of catalytic zinc sites. Hu-
mans require a daily intake of 15 mg to maintain normal zinc
concentrations (Bryce-Smith 1989), and zinc is involved in a
wide range of functions that are essential for both physical and
mental health; zinc is important to physiological functions in
the bone, kidney and brain (Sly et al. 1983). Zinc deficiency
can cause retardation, cessation of growth, impaired wound
healing, hair loss or defects leading to reproductive failure.
Zinc supplementation has successfully been used as a treat-
ment of many illnesses and disorders, including dwarfism,
sexual immaturity, acrodermatitis enteropathica (inflamma-
tion of the skin and the small intestine), anorexia nervosa and
bulimia nervosa (Bryce-Smith 1989). An improved under-
standing of catalytic zinc sites is vital to an improved under-
standing of the role of zinc in the whole organism. Further-
more, catalytic zinc sites provide convenient targets for drugs
because a wide range of functional groups (i.e., sulfonamides or
hydroxamates) can coordinate directly to the metal, displacing
the zinc-water in the active site and inhibiting the enzyme.
This has been exploited in the use of topical CA inhibitors to
lower intraocular pressure in patients with glaucoma (Lippa
1991) and may provide a route to the development of novel
antibiotics by inhibition of key enzymes in the lipid A bio- FIGURE 2 Example of cocatalytic zinc site: phospholipase C
synthetic pathway (Wyckoff et al. 1998). (Hough et al. 1989). In phospholipase C, as in nuclease P1, the back-
The active site features that delineate the catalytic role of bone amino and carbonyl groups of N-terminal Trp1 coordinate Zn2.
1442S SUPPLEMENT

SCHEME 4

bottom of the cavity. These ligands form an extensive hydro-


gen bond network with other residues: N␦-H of H94 is a
hydrogen bond donor to O⑀1 of Q92, N␦-H of H96 is a hydro-
gen bond donor to the backbone carbonyl oxygen of N244,
N⑀-H of H119 is a hydrogen bond donor to O⑀1 of E117 and the
zinc-bound water is a hydrogen bond donor to O␥ of Thr199,
which in turn is a hydrogen bond donor to O⑀1 of Glu106 (Fig.
4). These hydrogen bond acceptors to the direct ligands are
called “indirect ligands.” One side of the active site cavity is
mainly composed of hydrophilic residues, whereas the other
side contains mostly hydrophobic residues. This hydrophobic
region is probably the substrate CO2 binding site, as indicated FIGURE 4 Active site structure of CA II (Hakansson et al. 1992).
by the structure of the complex of the enzyme with bicarbon-

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ate and formate (Hakansson et al. 1992, Hakansson and
Wehnert 1992) and site-directed mutagenesis experiments
combined with FTIR spectroscopy (Fierke et al. 1991, Krebs spinach CA (Bracey et al. 1994). Phylogenetic analysis of
and Fierke 1993, Krebs et al. 1993a, 1993b). His64 is located at protein sequences and those deduced from cDNA sequences
the entrance of one side of the cavity; an ordered water chain have identified three independent CA gene families, desig-
connecting this side chain to zinc-bound water may function nated as ␣-CA, ␤-CA and ␥-CA. All animal CAs belong to
as the H⫹ transfer pathway (Hakansson et al. 1992, Jackman the ␣-CA family; the ␤-CA family is primarily composed of
et al. 1996) (Fig. 4). Based on the crystal structure of the CAs in plant chloroplasts and some eubacteria; and CAs
enzyme complexed with bisulfite, a catalytic mechanism has isolated from archaebacterium belong to the ␥-CA family
been proposed, involving a transient pentacoordinate zinc ion (Hewett-Emmett and Tashian 1996). ␣-CA is a monomer,
(Fig. 5) (Hakansson et al. 1992). and ␤-CA and ␥-CA are multimers. Although there is little
Other families of CA. Although the CAs from the seven primary sequence similarity between the three families, many
known isozymes and across a wide variety of species show a of the important active site residues are conserved, and all
high degree of homology, two CAs have been discovered that three CA families contain zinc at the active site (Bracey et al.
differ significantly from mammalian CA II: a CA from the 1994, Kisker et al. 1996, Lindskog 1997, Vallee and Galdes
archaeon Methanosarcina thermophila (Kisker et al. 1996) and 1984). The CA from M. thermophila is a representative of the
␥-CA family, of which the structure has been solved. The

FIGURE 3 The ribbon diagram of the structure of human CA II


(Hakansson et al. 1992). The zinc-liganding side chains H94, H96 and FIGURE 5 Proposed mechanism of the conversion of CO2 to
H119 are shown as ball-and-stick models. bicarbonate catalyzed by CA II (adapted from Hakansson et al. 1992).
FUNCTION AND MECHANISM OF ZINC METALLOENZYMES 1443S

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

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tigated using the complementary techniques of molecular bi- where the indirect ligands have been eliminated as hydrogen
ology, enzymology and structural biology. These studies high- bond acceptors by the substitution of alanine for Q92, E117 or
light the functional importance of the nature of the zinc T199 decreases ⬇ 10-fold per substituted indirect ligand (Kiefer
ligands, the structure of the active site hydrogen bond net- et al. 1995). This loss of zinc-binding affinity is not due to the
works and the hydrophobic residues surrounding the zinc site total loss of a hydrogen bond; compensatory hydrogen bonds
(Huang et al. 1996, Kiefer and Fierke 1994, Kiefer et al. 1995). are formed to either water or alternative amino acid side
The direct ligands. In CA II, and probably in all catalytic chains (Lesburg and Christianson 1995, Xue et al. 1993). In
zinc sites, the protein scaffolding modulates the chemical each case, the hydrogen bond to the direct ligand is weakened,
properties of the zinc ion and zinc-bound solvent. Specifically, due to either the entropic cost of sequestering a solvent mol-
the protein plays a critical role in lowering the pKa of zinc-
ecule into the hydrophobic active site (Fersht 1987) or the
bound water to 6.8 from 10 in solution (Woolley 1975) and
nonoptimal hydrogen bonding stereochemistry. The weaken-
increasing the second-order rate constant for CO2 hydration
ing of these hydrogen bonds should increase the mobility of
by ⬎ 104-fold (Coleman 1984). Structure-based dissection of
the direct ligands in the zinc-binding site of CA II (Alexander the direct ligands, and hence the role of the indirect ligands is
et al. 1993, Ippolito et al. 1995, Ippolito and Christianson to preorganize the histidines for optimal zinc coordination and
1994, Kiefer and Fierke 1994, Kiefer et al. 1993a, 1993b, Xue avidity. Indications of the indirect ligands acting to “fine-
et al. 1994) suggests that the electrostatic environment of the tune” the electrostatic environment of the zinc ion were also
zinc ion is a principal feature governing the chemical proper- seen.
ties of the metal site. In particular, the substitution of nega- Not only are all three histidines preordered by a hydrogen
tively charged groups for neutral histidine ligands significantly bonding network (Kiefer et al. 1995) but also the zinc-water
increases the pKa of the zinc-bound water (⬎ 1.4 pH units) (wat263) is preordered by its hydrogen bond to T199, which is
while simultaneously decreasing its CO2 hydration activity (⬎ in turn hydrogen-bonded to E106. The water-to-T199 hydrogen
1000-fold) (Kiefer and Fierke 1994). In addition, the affinity of bond seems to be a high energy interaction (Krebs et al.
sulfonamide inhibitors, in which the sulfonamide anionic ni- 1993a), and disruption of this bond is likely to incur an
trogen displaces the zinc-water to directly coordinate zinc energetic penalty. Even in the apo-CA II structure, the water
(Vidgren et al. 1993), decreases ⬎ 104-fold (Kiefer and Fierke is present in a similar position as in the zinc structure (Ha-
1994). These data suggest that the neutral ligand field in CA kansson et al. 1992), although it is pulled 0.3Å further from
is essential for high affinity coordination of anions and effi- the T199 by an apparent attraction to the nitrogens on H119
cient catalysis of CO2 hydration. and H94. The importance of preordering the catalytic water in
To further test this hypothesis, two neutral amino acid encouraging a tetrahedral binding site is underlined by two
substitutions, asparagine or glutamine, were substituted for the branches of evidence; when the threonine is eliminated, by
histidine zinc ligands. The slight increase in the pKa of zinc- making a T199A substitution, the zinc ion is found to have two
bound water and the high affinity for sulfonamide inhibitors of solvent ligands (Xue et al. 1993) rather than only one and the
these carboxamide CA II variants indicate that the positive variant shows enhanced bicarbonate binding as well (Liang et
charge on the zinc ion is crucial for stabilizing bound anions at al. 1993). Both the altered position of the zinc-water, and the
the active site of CA II (Lesburg et al. 1997). Furthermore, in increased product binding may be expected to reduce activity
each case, the activity of the asparagine or glutamine substi- in the T199A variant, and the CO2 hydration activity and the
tution was higher than the respective aspartate or glutamate esterase activity of the variant are in fact reduced ⬇ 100-fold
substitution, suggesting that the net positive charge at the compared with the wild-type enzyme. Furthermore, the pKa of
active site is important for stabilizing the catalytic transition the catalytic water is now ⬇ 1 pH unit higher than in the
state. However, the activity of the CA II variants with car- wild-type enzyme (Liang et al. 1993), indicating that T199 not
boxamide side chains coordinating zinc decreased compared only preorders the zinc-water but also assists in polarizing the
with the wild-type His3 metal polyhedron in each case. This water molecule. Altogether, these data indicate that the H-
activity loss in the carboxamide CA II variants is mainly bonding network to zinc-water is crucial for catalytic activity
caused by structural effects; the X-ray structures of these CA II and must be included in enzyme modeling or design attempts.
1444S SUPPLEMENT

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