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Supplement

Function and Mechanism of Zinc Metalloenzymes¹

Duke University Medical Center, Durham, NC 27710

²To whom correspondence should be addressed at Chemistry Department, University of Michigan, 930 N. University, Ann Arbor, MI 48109.

	ABSTRACT

TOP ABSTRACT INTRODUCTION Properties of zinc Application of knowledge REFERENCES

 
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 understanding 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.

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Properties of zinc Application of knowledge REFERENCES

 
Zinc was first shown to be required for the growth of the mold Aspergillus niger by Raulin in 1869. Since then, zinc has been demonstrated to be essential for the growth, development and differentiation of all types of life, including microorganisms, plants and animals (Vallee 1986 ). After iron, zinc is the second most abundant trace metal in the human body; an average 70-kg adult human contains 2.3 g of zinc (McCance and Widdowson 1942 ). The first zinc metalloenzyme, carbonic anhydrase II (CA³ II, EC 4.2.1.1), was discovered in 1940 by Keilin and Mann. Since then, > 300 zinc enzymes covering all six classes of enzymes and in different species of all phyla have been discovered (Christianson 1991 , Coleman 1992 , Vallee and Auld 1990a ). In most cases, the zinc ion is an essential cofactor for the observed biological function of these metalloenzymes. Furthermore, the biological functions of zinc, which are versatile and observed in many tissues, are most often associated with proteins.

	Properties of zinc

TOP ABSTRACT INTRODUCTION Properties of zinc Application of knowledge REFERENCES

 
The inherent chemical potential and reactivity of zinc are not exceptional compared with those of other metals (Cotton and Wilkinson 1988 ). However, unlike other first-row transition metals (e.g., Sc²⁺, Ti²⁺, V²⁺, Cr²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺ and Cu²⁺), the zinc ion (Zn²⁺) contains a filled d orbital (d¹⁰) and therefore does not participate in redox reactions but rather functions as a Lewis acid to accept a pair of electrons (Williams 1987 ). This lack of redox activity makes Zn²⁺ a stable ion in a biological medium whose potential is in constant flux. Therefore, the zinc ion is an ideal metal cofactor for reactions that require a redox-stable ion to function as a Lewis acid–type catalyst (Butler 1998 ), such as proteolysis and the hydration of carbon dioxide. Furthermore, due to the filled d-shell orbitals, Zn²⁺ has a ligand-field stabilization energy of 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 accessible coordination geometries can be used by zinc metalloenzymes to alter the reactivity of the metal ion and may be an important factor in the ability of Zn²⁺ to catalyze chemical transformations accompanied by changes in the metal coordination geometry. Nevertheless, in all zinc metalloenzymes studied to date, the binding geometry observed most often is a slightly distorted tetrahedral (Scheme 1^*) with the metal ion coordinating three or four protein side chains. However, five-coordinate distorted trigonal bipyramidal geometry has been observed in the metal sites of Zn-substituted astacin (EC 3.4.24.21) (Bode et al. 1992 ), two CA II (EC 4.2.1.1) mutants (H₁₁₉D and H₉₄D) (Ippolito and Christianson 1994 , Kiefer et al. 1993a ), the bimetal sites of purple acid phosphatase (EC 4.2.1.1) (Klabunde et al. 1996 ) and the trimetal sites of phospholipase C (EC 3.1.4.3) (Hough et al. 1989 ). In addition, five-coordinate geometry has been suggested for the reaction intermediate in CA (Christianson and Fierke 1996 , Lindskog 1997 ) and carboxypeptidase A (EC 3.4.17.1) (Christianson and Lipscomb 1989 ). A final important property of Zn²⁺ that makes it well suited as a catalytic cofactor is that ligand exchange is rapid (Cotton and Wilkinson 1988 ), allowing for the rapid product dissociation required for efficient turnover.

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Early examinations of the coordination preferences of zinc ions concluded that zinc preferentially bound to sulfur ligands due to the predominance of zinc sulfides in zinc ores, e.g., wurtzite, a hexagonal ZnS array containing zinc in a distorted tetrahedral coordination geometry, and zinc blende, a cubic ZnS array containing zinc in a perfect tetrahedral coordination geometry (Cotton and Wilkinson 1988 , Vallee and Auld 1990a ). However, after an examination of a variety of small molecule coordination complexes in solution, Pearson (1963 ) classified zinc as a "borderline" metal, meaning that Zn²⁺ does not consistently act either "hard" (not very polarizable) or "soft" (highly polarizable) and does not have a strong preference for coordinating with either oxygen, nitrogen or sulfur atoms. In protein zinc-binding sites, the zinc ion is coordinated by different combinations of protein side chains, including the nitrogen of histidine, the oxygen of aspartate or glutamate and the sulfur of cysteine; among these, histidine is most commonly observed, followed by cysteine (Gregory et al. 1993 ) (Table 1 ). Other, much more rarely observed ligands include the hydroxyl of tyrosine, the carbonyl oxygen of the protein backbone and the carbonyl oxygen of either asparagine or glutamine. The varied ligands and coordination geometries in zinc metalloenzymes result in zinc-binding sites with a broad range of stability constants, reactivities and functions.

A catalytic zinc ion is located at the active site of an enzyme, where it participates directly in the catalytic mechanism, interacting with the substrate molecules undergoing reaction. A unique feature for a catalytic zinc site is the existence of an open coordination sphere; that is, the 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 polyhedron 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 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 the zinc hydrolases and lyases, such as the zinc proteases and CAs, the zinc ion serves as a powerful electrophilic catalyst by providing all or a combination of the following: (1) an activated water molecule for nucleophilic attack, (2) polarization of the carbonyl of the scissile bond and (3) stabilization of the negative charge in the transition state (Christianson and Cox, 1999 , Lovejoy et al. 1994 , Silverman and Lindskog 1988 , Vallee and Galdes 1984 ).

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The X-ray structures of catalytic zinc enzymes from four of the six classes of enzymes (oxidoreductases, transferases, hydrolases and lyases) have been determined, and they define the features of catalytic zinc-binding sites. Unlike the structural sites, the metal ion in catalytic sites is generally coordinated to the side chain of three amino acid residues, a combination of histidine, glutamate, aspartate and cysteine, and a solvent molecule completes the tetrahedral coordination sphere (Table 1) (for reviews, see Christianson 1991 , Jernigan et al. 1994 , Vallee and Auld 1990b ). However, the zinc polyhedra of adenosine deaminase (EC 3.5.4.4) (Wilson and Quiocho 1993 , Wilson et al. 1991 ) and astacin (EC 3.4.24.21) (Gomisruth et al. 1993 ) are composed of four amino acid side chains and a solvent molecule with a trigonal bipyramidal geometry.

In catalytic zinc sites, histidine is the most frequently observed ligand, distantly followed by glutamic acid and then aspartic acid and cysteine. In non–coenzyme-dependent zinc enzymes, a short spacer with a rigid arrangement of one to three amino acids intervening between the first two ligands, L₁ and L₂ (Table 1) , may constitute a nucleus for the zinc-binding site (i.e., CA site, Fig. 1 ). The third ligand, L₃, is separated from L₂ by a longer spacer whose length varies greatly (5–200 amino acids) and may be responsible for the spatial formation of the active site, allowing some flexibility to the coordination sphere.

View larger version (48K): [in this window] [in a new window]   Figure 1. The zinc-binding site of CA II. The first two ligands, H94 and H96, are on the same stand of the ß-sheet. The third ligand, H119, is on the neighboring strand of the ß-sheet.

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A feature common to all zinc sites is that the metal ion is surrounded by a shell of hydrophilic groups that is embedded within a larger shell of hydrophobic groups (Yamashita et al. 1990 ). In addition, the amino acid side chains serving as zinc ligands in these structures often make hydrogen bond contacts with other residues, perhaps to preorder the metal ion binding site and lower the entropic cost of binding the metal ion (Christianson 1991 ). These interactions between metal ion and ligand have been proposed to orient the metal ligands, enhance the electrostatic interaction between metal and ligand and modulate the zinc-water pK_a (Argos et al. 1978 , Christianson 1991 ).

Interestingly, the catalytic metal in two zinc metalloproteins is believed to have a central role in regulation of enzyme activity. The first example is stromelysin (EC 3.4.24.17), which is produced as an inactive proenzyme and activated by proteolysis of 80 amino acids from the N terminus (Van Wart and Birkedal-Hansen 1990 ). This activation involves the replacement of a thiolate zinc ligand with a water molecule, yielding the catalytically active His₃H₂O zinc site (Gooley et al. 1993 , Holz et al. 1992 , Salowe et al. 1992 , Van Wart and Birkedal-Hansen 1990 ).

The second case is the Escherichia coli Ada protein, a DNA repair protein with an unusual zinc site (Myers et al. 1992 ). The tetrahedral Cys₄ zinc polyhedron in the N-terminal domain of Ada is important to stabilize the tertiary fold. However, in addition, this zinc site catalyzes the direct, irreversible transfer of a methyl group from the S_p diastereomer of DNA methylphosphotriester to the sulfur of Cys₆₉ in the coordination sphere (Myers et al. 1993a ). This conversion of the thiolate metal ligand to a more weakly bound thioether ligand on methyl transfer has been proposed to propel structural changes that reveal the sequence-specific DNA binding conformation of the protein (Myers et al. 1993a ). In this conformation, Ada functions as a transcription factor, inducing genes that confer resistance to methylating agents. The proposed role of the zinc ion in Ada, coordinating the cysteine thiolate to lower the pK_a and enhance the reactivity of this group (Matthews and Goulding 1997 , Myers et al. 1993 ), is similar to the role being proposed for zinc in several enzymes that catalyze S-alkylation reactions, such as cobalamine-dependent methionine synthase (EC 2.1.1.13), cobalamine-independent methionine synthase (EC 2.1.1.14), methanol:coenzyme M methyltransferase (EC 2.1.1.86) and protein farnesyltransferase (EC 2.5.1.21) (Gonzalez et al. 1996 , Goulding and Matthews 1997 , Hightower et al. 1998 , Huang et al. 1997 , LeClerc and Grahame 1996 ). This class of zinc metalloproteins may indicate a new catalytic function of the zinc ion: to enhance the nucleophilicity of a thiol group at neutral pH (Hightower and Fierke 1999, Matthews and Goulding 1997 ).

Cocatalytic zinc sites.

In multimetal enzymes, the two or more zinc (or other metal) atoms may operate in concert to enhance catalysis. A class of catalytic zinc sites, called cocatalytic zinc sites, has been defined in which two or more zinc atoms are in close proximity to one another (Vallee and Auld 1992a , 1992b , 1993b ). This group of enzymes includes alkaline phosphatase (with two zinc ions and one magnesium ion), phospholipase C (three zinc ions), nuclease P1 (EC 3.1.30.1; three zinc ions) and leucine aminopeptidase (two zinc ions) (Vallee and Auld 1993b ). A representative structure (phospholipase C) is shown in Figure 2 . The first zinc ion, designated as the catalytic zinc (Zn₁ in Fig. 2 ), contains a bound water that is essential for catalysis and has an His₂Glu metal polyhedron similar to those found in other single catalytic zinc sites (Table 1) . However, the second zinc (Zn₂) and the third metal (Zn₃/Mg) ion sites may have unusual ligands such as the oxygen of serine or threonine or the nitrogen of the N-terminal amino group. An additional distinctive feature is the existence of one or more bridging ligands (either aspartate or water or both) between the second zinc ion (Zn₂) and the third zinc ion (Zn₃) or magnesium ion (Zn₃/Mg), which often leads to pentacoordinate geometry. The distance between the catalytic zinc ion (Zn₁) and the second zinc ion (Zn₂), at 4–5 Å, is shorter than that between the catalytic zinc ion and the third zinc ion (Zn₃), at 6–7 Å (Fig. 2) . For phosphate ester hydrolyzing enzymes, the product phosphate interacts with all three metals, displacing the weak unusual ligands in the second and third zinc sites to facilitate catalysis (Vallee and Auld 1993b ). These sites are termed "cocatalytic" because all three metals play crucial roles in catalysis despite only the zinc activating the attacking water being termed "catalytic."

View larger version (16K): [in this window] [in a new window]   Figure 2. Example of cocatalytic zinc site: phospholipase C (Hough et al. 1989 ). In phospholipase C, as in nuclease P1, the backbone amino and carbonyl groups of N-terminal Trp1 coordinate Zn2.

In structural zinc sites, the metal ion is coordinated by four amino acid side chains, usually in a tetrahedral geometry, so that solvent is excluded as an inner sphere ligand (for reviews, see Vallee and Auld 1990b , Vallee et al. 1991 ). Cysteine is by far the ligand observed most frequently in these sites, with histidine also being present in many cases and aspartate being present in one case (Lovejoy et al. 1994 , Spurlino et al. 1994 ). In contrast to catalytic zinc sites, these sites contain no regular pattern of spacer length between the protein zinc ligands, and the ligands can be located on a flexible loop rather than in a rigid secondary structure. The high stability constants of these tetradentate zinc complexes ensure both local and overall structural stability similar to that provided by disulfides (Vallee and Auld 1990b ). This enables proteins containing structural zinc atoms to perform a wide range of functions.

Importance of further study of catalytic zinc sites.

Humans 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 treatment of many illnesses and disorders, including dwarfism, sexual immaturity, acrodermatitis enteropathica (inflammation of the skin and the small intestine), anorexia nervosa and bulimia nervosa (Bryce-Smith 1989 ). An improved understanding of catalytic zinc sites is vital to an improved understanding of the role of zinc in the whole organism. Furthermore, 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 biosynthetic pathway (Wyckoff et al. 1998 ).

The active site features that delineate the catalytic role of 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 Vincent 1984 ). CA II plays a variety of physiological roles, including promoting CO₂ exchange in the erythrocytes, kidney and lung; contributing to acid-base homeostasis; and promoting 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 additional 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⁺ 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 Lindskog 1988 ).

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The high resolution x-ray crystal structure of human CA II was first solved in 1972 (Liljas et al. 1972 ) and further refined to 2 Å (Eriksson et al. 1988 ) and then to 1.54 Å (Hakansson et al. 1992 ) (Fig. 3 ). The active site cavity is 15 Å wide at the entrance and 15 Å in depth. The zinc ion is coordinated to three histidine residues, N of H₉₄, N of H₉₆ and N of H₁₁₉, and a water molecule in a tetrahedral manner at the bottom of the cavity. These ligands form an extensive hydrogen bond network with other residues: N-H of H₉₄ is a hydrogen bond donor to O1 of Q₉₂, N-H of H₉₆ is a hydrogen bond donor to the backbone carbonyl oxygen of N₂₄₄, N-H of H₁₁₉ is a hydrogen bond donor to O1 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 O1 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 by the structure of the complex of the enzyme with bicarbonate and formate (Hakansson et al. 1992 , Hakansson and Wehnert 1992 ) and site-directed mutagenesis experiments combined with FTIR spectroscopy (Fierke et al. 1991 , Krebs and Fierke 1993 , Krebs et al. 1993a , 1993b ). His₆₄ is located at the entrance of one side of the cavity; an ordered water chain connecting this side chain to zinc-bound water may function as the H⁺ transfer pathway (Hakansson et al. 1992 , Jackman et al. 1996 ) (Fig. 4) . Based on the crystal structure of the enzyme complexed with bisulfite, a catalytic mechanism has been proposed, involving a transient pentacoordinate zinc ion (Fig. 5 ) (Hakansson et al. 1992 ).

View larger version (81K): [in this window] [in a new window]   Figure 3. The ribbon diagram of the structure of human CA II (Hakansson et al. 1992 ). The zinc-liganding side chains H94, H96 and H119 are shown as ball-and-stick models.

View larger version (15K): [in this window] [in a new window]   Figure 4. Active site structure of CA II (Hakansson et al. 1992 ).

View larger version (15K): [in this window] [in a new window]   Figure 5. Proposed mechanism of the conversion of CO2 to bicarbonate catalyzed by CA II (adapted from Hakansson et al. 1992 ).

Although the CAs from the seven known isozymes and across a wide variety of species show a high degree of homology, two CAs have been discovered that differ significantly from mammalian CA II: a CA from the archaeon Methanosarcina thermophila (Kisker et al. 1996 ) and spinach CA (Bracey et al. 1994 ). Phylogenetic analysis of protein sequences and those deduced from cDNA sequences have identified three independent CA gene families, designated as -CA, ß-CA and -CA. All animal CAs belong to the -CA family; the ß-CA family is primarily composed of CAs in plant chloroplasts and some eubacteria; and CAs isolated from archaebacterium belong to the -CA family (Hewett-Emmett and Tashian 1996 ). -CA is a monomer, and ß-CA and -CA are multimers. Although there is little primary sequence similarity between the three families, many of the important active site residues are conserved, and all three CA families contain zinc at the active site (Bracey et al. 1994 , Kisker et al. 1996 , Lindskog 1997 , Vallee and Galdes 1984 ). The CA from M. thermophila is a representative of the -CA family, of which the structure has been solved. The active form of the enzyme is a trimer of left-handed ß-helices, and the zinc-containing active sites are located at the interfaces between two monomers, yet the active site itself is strikingly similar to that of CA II. The zinc-binding site is made up of His₈₁ and His₁₂₂ from one monomer and His₁₁₇ from the neighboring monomer along with a coordinated water. Similarly, the shell of "indirect ligands" is also found; the hydrogen bond acceptors of CA II, Q₉₂, N₂₄₄ and E₁₁₇, have their analogs in the CA of M. thermophila, D₆₁, G₁₂₃ and D₇₆, respectively. In the ß-CA family, no high resolution structure is currently available. However, because sulfonamides inhibit plant-type CAs (Pocker and Ng 1974 ) as they do in mammalian CAs, it is presumed that the zinc bound to the plant enzyme is also catalytic. Furthermore, the zinc polyhedron of spinach CA, identified by EXAFS (extended X-ray absorption fine structure) and mutagenesis studies, reveals a tetracoordinate His₁Cys₂Wat₁ zinc site (Bracey et al. 1994 , Rowlett 1984 ), supporting the suggestion that the zinc is catalytic.

Investigation of CA II.

The determinants of metal affinity and catalysis in the zinc-binding site of CA have been investigated using the complementary techniques of molecular biology, enzymology and structural biology. These studies highlight the functional importance of the nature of the zinc ligands, the structure of the active site hydrogen bond networks and the hydrophobic residues surrounding the zinc site (Huang et al. 1996 , Kiefer and Fierke 1994 , Kiefer et al. 1995 ).

The direct ligands.

In CA II, and probably in all catalytic zinc sites, the protein scaffolding modulates the chemical properties of the zinc ion and zinc-bound solvent. Specifically, the protein plays a critical role in lowering the pK_a of zinc-bound water to 6.8 from 10 in solution (Woolley 1975 ) and increasing the second-order rate constant for CO₂ hydration by > 10⁴-fold (Coleman 1984 ). Structure-based dissection of the direct ligands in the zinc-binding site of CA II (Alexander et al. 1993 , Ippolito et al. 1995 , Ippolito and Christianson 1994 , Kiefer and Fierke 1994 , Kiefer et al. 1993a , 1993b , Xue et al. 1994 ) suggests that the electrostatic environment of the zinc ion is a principal feature governing the chemical properties of the metal site. In particular, the substitution of negatively charged groups for neutral histidine ligands significantly increases the pK_a of the zinc-bound water (> 1.4 pH units) while simultaneously decreasing its CO₂ hydration activity (> 1000-fold) (Kiefer and Fierke 1994 ). In addition, the affinity of sulfonamide inhibitors, in which the sulfonamide anionic nitrogen displaces the zinc-water to directly coordinate zinc (Vidgren et al. 1993 ), decreases > 10⁴-fold (Kiefer and Fierke 1994 ). These data suggest that the neutral ligand field in CA is essential for high affinity coordination of anions and efficient catalysis of CO₂ hydration.

To further test this hypothesis, two neutral amino acid substitutions, asparagine or glutamine, were substituted for the histidine zinc ligands. The slight increase in the pK_a of zinc-bound water and the high affinity for sulfonamide inhibitors of these carboxamide CA II variants indicate that the positive charge on the zinc ion is crucial for stabilizing bound anions at the active site of CA II (Lesburg et al. 1997 ). Furthermore, in each case, the activity of the asparagine or glutamine substitution was higher than the respective aspartate or glutamate substitution, suggesting that the net positive charge at the active site is important for stabilizing the catalytic transition state. However, the activity of the CA II variants with carboxamide side chains coordinating zinc decreased compared with the wild-type His₃ metal polyhedron in each case. This activity loss in the carboxamide CA II variants is mainly caused by structural effects; the X-ray structures of these CA II variants reveal that either the zinc-binding geometry is changed from tetrahedral to trigonal bipyramidal (H₉₄N CA II and H₁₁₉N CA II) or the position of the zinc-bound water is moved (H₁₁₉Q CA II) (Lesburg et al. 1997 ). The bulky histidine ligands (especially H₁₁₉) may play a role in disfavoring higher coordination numbers, and therefore stabilizing a low coordination number, for the active site zinc ion of native CA II. This decreased coordination number should both depress the pK_a of zinc-bound solvent and increase its reactivity (Bertini et al. 1990 ). Furthermore, the metal affinity of the variants with carboxamide ligands is significantly compromised (Lesburg et al. 1997 ). Taken together, these data indicate that the neutral histidine ligands of the zinc-binding site optimize the electrostatic environment of the active site to maintain high catalytic activity and high zinc affinity in CA II.

The indirect ligands.

Structure-based dissection of the indirect ligands of CA II (Huang et al. 1996 , Kiefer et al. 1995 , Lesburg and Christianson 1995 ) implicated these residues in "fine tuning" the electrostatic environment of the zinc ion and enhancing zinc-binding affinity. The zinc affinity of variants where the indirect ligands have been eliminated as hydrogen bond acceptors by the substitution of alanine for Q₉₂, E₁₁₇ or T₁₉₉ decreases 10-fold per substituted indirect ligand (Kiefer et al. 1995 ). This loss of zinc-binding affinity is not due to the total loss of a hydrogen bond; compensatory hydrogen bonds are formed to either water or alternative amino acid side chains (Lesburg and Christianson 1995 , Xue et al. 1993 ). In each case, the hydrogen bond to the direct ligand is weakened, due to either the entropic cost of sequestering a solvent molecule into the hydrophobic active site (Fersht 1987 ) or the nonoptimal hydrogen bonding stereochemistry. The weakening of these hydrogen bonds should increase the mobility of the direct ligands, and hence the role of the indirect ligands is to preorganize the histidines for optimal zinc coordination and avidity. Indications of the indirect ligands acting to "fine-tune" the electrostatic environment of the zinc ion were also seen.

Not only are all three histidines preordered by a hydrogen bonding network (Kiefer et al. 1995 ) but also the zinc-water (wat₂₆₃) is preordered by its hydrogen bond to T₁₉₉, which is in turn hydrogen-bonded to E₁₀₆. The water-to-T₁₉₉ hydrogen bond seems to be a high energy interaction (Krebs et al. 1993a ), and disruption of this bond is likely to incur an energetic penalty. Even in the apo-CA II structure, the water is present in a similar position as in the zinc structure (Hakansson et al. 1992 ), although it is pulled 0.3Å further from the T₁₉₉ by an apparent attraction to the nitrogens on H₁₁₉ and H₉₄. The importance of preordering the catalytic water in encouraging a tetrahedral binding site is underlined by two branches of evidence; when the threonine is eliminated, by making a T₁₉₉A substitution, the zinc ion is found to have two solvent ligands (Xue et al. 1993 ) rather than only one and the variant shows enhanced bicarbonate binding as well (Liang et al. 1993 ). Both the altered position of the zinc-water, and the increased product binding may be expected to reduce activity in the T₁₉₉A variant, and the CO₂ hydration activity and the esterase activity of the variant are in fact reduced 100-fold compared with the wild-type enzyme. Furthermore, the pK_a of the catalytic water is now 1 pH unit higher than in the wild-type enzyme (Liang et al. 1993 ), indicating that T₁₉₉ not only preorders the zinc-water but also assists in polarizing the water molecule. Altogether, these data indicate that the H-bonding network to zinc-water is crucial for catalytic activity and must be included in enzyme modeling or design attempts.

The hydrophobic core.

The role of highly conserved aromatic residues near the zinc-binding site of CA II in affecting metal ion binding has also been examined. Residues F₉₃, F₉₅ and W₉₇ are located along the ß-strand containing the direct ligands H₉₄ and H₉₆ and contribute to the high zinc affinity and slow zinc dissociation rate of CA II (Hunt and Fierke 1997 ). Variants in which smaller amino acids are substituted at these positions result in up to eightfold reductions in Zn/Co specificity and > 10⁴-fold reductions in Zn/Cu specificity (Hunt et al. 1999 ). Similar changes in metal specificity are observed when wild-type CA II is partially unfolded by incubation with guanidine hydrochloride, suggesting that the changes in metal binding properties are due to increased flexibility of the protein structure in the metal binding site (Hunt et al. 1999 ). These data reveal one of the structural features a protein may use to optimize binding specificity for a catalytic metal ion; the high stability of the protein structure surrounding the direct ligands. Knowledge of the structural factors that lead to high metal ion specificity will aid in the design of metal ion biosensors.

	Application of knowledge

TOP ABSTRACT INTRODUCTION Properties of zinc Application of knowledge REFERENCES

 
An understanding of naturally occurring metal ion binding sites, in particular the factors governing specificity and avidity, will also aid in creating de novo metal-binding proteins and in designing new metal sites in existing proteins. Such metal-binding sites could be designed to act as metal-activated switches for control of activity (McGrath et al. 1993 ), to add stability or to serve as metal ion biosensors (Thompson and Jones 1993 ). The use of modified metalloproteins as sensors has several advantages over current analytical techniques. The use of biomolecules allows high selectivity in the recognition of analytes, such as metal ions, in complex natural solutions, e.g., seawater or blood, and the combination of this property with a covalently attached fluorescent probe has shown great promise as an indicator system that may in the future replace current techniques of measuring very low concentrations of metal ions (Thompson et al. 1996 ).

	FOOTNOTES

 
¹ Presented at the international workshop "Zinc and Health: Current Status and Future Directions," held at the National Institutes of Health in Bethesda, MD, on November 4–5, 1998. This workshop was organized by the Office of Dietary Supplements, NIH and cosponsored with the American Dietetic Association, the American Society for Clinical Nutrition, the Centers for Disease Control and Prevention, Department of Defense, Food and Drug Administration/Center for Food Safety and Applied Nutrition and seven Institutes, Centers and Offices of the NIH (Fogarty International Center, National Institute on Aging, National Institute of Dental and Craniofacial Research, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute on Drug Abuse, National Institute of General Medical Science and the Office of Research on Women’s Health). Published as a supplement to The Journal of Nutrition. Guest editors for this publication were Michael Hambidge, University of Colorado Health Science Center, Denver; Robert Cousins, University of Florida, Gainesville; Rebecca Costello, Office of Dietary Supplements, NIH, Bethesda, MD; and session chair, Craig McClain, University of Kentucky, Lexington.

³ Abbbreviation used: CA, carbonic anhydrase.

	REFERENCES

TOP ABSTRACT INTRODUCTION Properties of zinc Application of knowledge REFERENCES

 

1. Alexander R. S., Kiefer L. L., Fierke C. A., Christianson D. W. Engineering the zinc binding site of human carbonic anhydrase II: structure of the His-94Cys apoenzyme in a new crystalline form. Biochemistry 1993;32:1510-1518[Medline]

2. Argos P., Garavito R. M., Eventoff W., Rossman M. G., Branden C. I. Similarities in active center geometries of zinc-containing enzymes, proteases and dehydrogenases. J. Mol. Biol. 1978;126:141-158[Medline]

3. Arnold F. H., Haymore B. L. Engineered metal-binding proteins: purification to protein folding. Science (Washington DC) 1991;252:1796-1797[Free Full Text]

4. Bertini I., Luchinat C., Rosi M., Sgamellotti A., Tarantelli F. pK_a of zinc-bound water and nucleophilicity of hydroxo-containing species: ab initio calculations on models for zinc enzymes. Inorg. Chem. 1990;29:1460-1463

5. Betts I., Xiang S., Short S. A., Wolfenden R., Carter C.W.J. Cytidine deaminase: the 2.3 Å crystal structure of an enzyme: transition-state analog complex. J. Mol. Biol. 1994;235:635-656[Medline]

6. Bode W., Gomisruth F. X., Huber R., Zwilling R., Stocker W. Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases. Nature (Lond.) 1992;358:164-167[Medline]

7. Bogin O., Peretz M., Burnstein Y. Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids. Prot. Sci. 1997;6:450-458[Abstract]

8. Bracey M. H., Christiansen J., Tovar P., Cramer S. P., Bartlett S. G. Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and EXAFS. Biochemistry 1994;33:13126-13131[Medline]

9. Bryce-Smith D. Zinc-deficiency: the neglected factor. Chem. Br. 1989;25:783-786

10. Butler A. Acquisition and utilization of transition metal ions by marine organisms. Science (Washington DC) 1998;281:207-209[Abstract/Free Full Text]

11. Carrell C. J., Carrell H. L., Erlebacher J., Glusker J. P. Structural aspects of metal ion-carboxylate interaction. J. Am. Chem. Soc. 1988;110:8651-8656

12. Cedergren-Zeppezauer E. S., Andersson I., Ottonello S., Bignetti E. X-ray analysis of structural changes induced by reduced nicotinamide adenine dinucleotide when bound to cysteine-46-carboxymethylated liver alcohol dehydrogenase. Biochemistry 1985;24:4000-4010[Medline]

13. Chakrabarti P. Geometry of interaction of metal ions with sulfur-containing ligands in protein structures. Biochemistry 1989;28:6081-6085[Medline]

14. Chakrabarti P. Geometry of interaction of metal ions with histidine residues in protein structures. Prot. Eng. 1990a;4:57-63[Abstract/Free Full Text]

15. Chakrabarti P. Interaction of metal ions with carboxylic and carboxamide groups in protein structures. Prot. Eng. 1990b;4:49-56[Abstract/Free Full Text]

16. Christianson D. W. The structural biology of zinc. Adv. Prot. Chem. 1991;42:281-335[Medline]

17. Christianson D. W., Cox J. D. Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes. Annu. Rev. Biochem. 1999;68:33-37[Medline]

18. Christianson D. W., Fierke C. A. Carbonic anhydrase: evolution of the zinc binding site by nature and by design. Acc. Chem. Res. 1996;29:331-339

19. Christianson D. W., Lipscomb W. N. Carboxypeptidase A. Acc. Chem. Res. 1989;22:62-69

20. Coleman J. E. Carbonic anhydrase: zinc and the mechanism of catalysis. Ann. N. Y. Acad. Sci. 1984;429:26-48[Medline]

21. Coleman J. E. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Richardson C. C. Abelson J. N. Meister A. Walsch C. T. eds. Annual Review of Biochemistry 1992:897-946 Annual reviews Inc Palo Alto, CA.

22. Cotton F. A., Wilkinson G. Advanced Inorganic Chemistry: A Comprehensive Text 5th ed., vol. 1. 1988 John Wiley & Sons New York.

23. Dideberg O., Charlier P., Dive G., Joris B., Frere J. M., Ghuysen J. M. Structure of a Zn²⁺-containing D-alanyl-D-alanine-cleaving carboxypeptidase at 2.5 Å resolution. Nature (Lond.) 1982;299:469-470[Medline]

24. Eriksson A. E., Jones T. A., Liljas A. Refined structure of human carbonic anhydrase II at 2.0 Å resolution. Proteins 1988;4:274-282[Medline]

25. Fersht A. R. The hydrogen-bond in molecular recognition. Trends Biochem. Sci. 1987;12:301-304

26. Fierke C. A., Calderone T. L., Krebs J. F. Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II. Biochemistry 1991;30:11054-11063[Medline]

27. Fuiji T., Hata Y., Oozeki M., Moriyama H., Wakagi T., Tanaka N., Oshima T. The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7. Biochemistry 1997;36:1505-1513[Medline]

28. Glusker J. P. Structural aspects of metal liganding to functional groups in proteins. Metalloproteins: Structural Aspects. Adv. Protein Chem 1991:1-76

29. Gomis-Ruth F. X., Stocker W., Huber R., Zwilling R., Bode W. Refined 1.8 Å x-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus-astacus l.: structure determination, refinement, molecular structure and comparison with thermolysin. J. Mol. Biol. 1993;229:945-968[Medline]

30. Gonzalez J. C., Peariso K., Penner-Hahn J. E., Matthews R. G. Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme. Biochemistry 1996;35:12228-12234[Medline]

31. Gooley P. R., Johnson B. A., Marcy A. I., Cuca G. C., Salowe S. P., Hagmann W. K., Esser C. K., Springer J. P. Secondary structure and zinc ligation of human recombinant short-form stromelysin by multidimensional heteronuclear NMR. Biochemistry 1993;32:13098-13108[Medline]

32. Goulding C. W., Matthews R. G. Cobalamin-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation. Biochemistry 1997;36:15749-15757[Medline]

33. Gregory D. S., Martin A.C.R., Cheetham J. C., Rees A. R. The prediction and characterization of metal binding sites in proteins. Prot. Eng. 1993;6:29-35[Abstract/Free Full Text]

34. Hakansson K., Carlsson M., Svensson L. A., Liljas A. Structure of native and apo carbonic anhydrase II and some of its anion-ligand complexes. J. Mol. Biol. 1992;227:1192-1204[Medline]

35. Hakansson K., Wehnert A. Structure of cobalt carbonic anhydrase complexed with bicarbonate. J. Mol. Biol. 1992;228:1212-1218[Medline]

36. Hellinga H. W. The construction of metal centers in proteins by rational design. Folding Design 1998;3:R1-R8[Medline]

37. Hellinga H. W., Caradonna J. P., Richards F. M. Construction of new ligand binding sites in proteins of known structure: II. Grafting of a buried transition metal binding site into Escherichia coli thioredoxin. J. Mol. Biol. 1991;222:787-803[Medline]

38. Hewett-Emmett D., Tashian R. E. Functional diversity, conservation and convergence in the evolution of the -, ß- and -carbonic anhydrase gene families. Mol. Phylogenet. Evol. 1996;5:50-77[Medline]

39. Hightower K. E., Huang C.C., Casey P. J., Fierke C. A. H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate. Biochemistry 1998;37:15555-15562[Medline]

40. Holz R. C., Salowe S. P., Smith C. K., Cuca G. C., Que L., Jr EXSAFS: evidence for a "cysteine switch" in the activation of prostromelysin. J. Am. Chem. Soc. 1992;114:9611-9614

41. Honzatko R. B., Crawford J. L., Monaco H. L., Ladner J. E., Edwards B.F.P., Evans D. R., Warren S. G., Wiley D. C., Ladner R. C., Lipscomb W. N. Crystal and molecular structures of native and CPP-liganded aspartate carbamoyltransferase from Escherichia coli. J. Mol. Biol. 1982;160:219-263[Medline]

42. Hough E., Hansen L. K., Birknes B., Jynge K., Hansen S., Hordvik A., Little C., Dodson E., Derewenda Z. High-resolution (1.5 Å) crystal structure of phospholipase C from Bacillus cereus. Nature (Lond.) 1989;338:357-360[Medline]

43. Huang C.-c., Casey P. J., Fierke C. A. Evidence for a catalytic role of zinc in protein farnesyltransferase: spectroscopy of Co²⁺-Ftase indicates metal coordination of the substrate thiolate. J. Biol. Chem. 1997;272:20-23[Abstract/Free Full Text]

44. Huang C.-c., Lesburg C. A., Kiefer L. L., Fierke C. A., Christianson D. W. Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibration kinetics in carbonic anhydrase II. Biochemistry 1996;35:3439-3446[Medline]

45. Huheey J. E., Keiter E. A., Keiter R. L. Inorganic Chemistry: Principles of Structure and Reactivity 4th ed., vol. 1 1993 Harper Collins College Publishers New York.

46. Hunt J. A., Ahmed M., Fierke C. A. Metal binding specificity in carbonic anhydrase is influenced by conserved hydrophobic core residues. Biochemistry 1999;38:9054-9062[Medline]

47. Hunt J. A., Fierke C. A. Selection of carbonic anhydrase variants displayed on phage: aromatic residues in zinc binding site enhance metal affinity and equilibration kinetics. J. Biol. Chem. 1997;272:20364-20372[Abstract/Free Full Text]

48. Ippolito J. A., Baird T. T., Jr, McGee S. A., Christianson D. W., Fierke C. A. Structure-assisted redesign of a protein-zinc binding site with femtomolar affinity. Proc. Natl. Acad. Sci. U.S.A. 1995;92:5017-5021[Abstract/Free Full Text]

49. Ippolito J. A., Christianson D. W. Structural consequences of redesigning a protein-zinc binding site. Biochemistry 1994;33:15241-15249[Medline]

50. Jackman J. E., Merz K. M., Jr, Fierke C. A. Disruption of the active site solvent network in carbonic anhydrase ii decreases the efficiency of proton transfer. Biochemistry 1996;35:16421-16428[Medline]

51. Jernigan R., Raghunathan G., Bahar I. Characterization of interactions and metal-ion binding-sites in proteins. Curr. Opin. Struct. Biol. 1994;4:256-263

52. Kiefer L. L., Fierke C. A. Functional characterization of human carbonic anhydrase II variants with altered zinc binding sites. Biochemistry 1994;33:15233-15240[Medline]

53. Kiefer L. L., Ippolito J. F., Fierke C. A., Christianson D. W. Redesigning the zinc binding site of human carbonic anhydrase II: structure of a His₂Asp-Zn²⁺ metal coordination polyhedron. J. Am. Chem. Soc. 1993a;115:12581-12582

54. Kiefer L. L., Krebs J. F., Paterno S. A., Fierke C. A. Engineering a cysteine ligand into the zinc binding site of human carbonic anhydrase II. Biochemistry 1993b;32:9896-9900[Medline]

55. Kiefer L. L., Paterno S. A., Fierke C. A. Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency. J. Am. Chem. Soc. 1995;117:6831-6837

56. Kim E. E., Wyckoff H. W. Reaction mechanism of alkaline phosphatase based on crystal structures: two-metal ion catalysis. J. Mol. Biol. 1991;218:449-464[Medline]

57. Kisker C., Schindelin H., Alber B. E., Ferry J. G., Rees D. C. A left-handed ß-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila. EMBO J 1996;15:2323-2330[Medline]

58. Klabunde T., Strater N., Frohlich R., Witzel H., Krebs B. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures. J. Mol. Biol. 1996;259:737-748[Medline]

59. Krebs J. F., Fierke C. A. Determinants of catalytic activity and stability of carbonic anhydrase ii as revealed by random mutagenesis. J. Biol. Chem. 1993;268:948-954[Abstract/Free Full Text]

60. Krebs J. F., Ippolito J. A., Christianson D. W., Fierke C. A. Structural and functional importance of a conserved hydrogen bond network in human carbonic anhydrase II. J. Biol. Chem. 1993a;268:27458-27466[Abstract/Free Full Text]

61. Krebs J. F., Rana F., Dluhy R. A., Fierke C. A. Kinetic and spectroscopic studies of hydrophilic amino acid substitutions in the hydrophobic pocket of human carbonic anhydrase II. Biochemistry 1993b;32:4496-4505[Medline]

62. LeClerc G. M., Grahame D. A. Methylcobamide:coenzyme M methyltransferase isozymes from Methanosarcina barkeri. J. Biol. Chem. 1996;271:18725-18731[Abstract/Free Full Text]

63. Lesburg C. A., Christianson D. W. X-ray crystallographic studies of engineered hydrogen bond networks in a protein-zinc binding site. J. Am. Chem. Soc. 1995;117:6838-6844

64. Lesburg C. A., Huang C.-c., Christianson D. W., Fierke C. A. Histidine carboxamide ligand substitutions in the zinc binding site of carbonic anhydrase II alter metal coordination geometry but retain catalytic activity. Biochemistry 1997;36:15780-15791[Medline]

65. Liang Z., Xue Y., Behravan G., Jonsson B.-H., Lindskog S. Importance of the conserved active-site residues Tyr7, Glu106 and Thr199 for the catalytic function of human carbonic anhydrase II. Eur. J. Biochem. 1993;211:821-827[Medline]

66. Liljas A., Kannan K. K., Bergsten P.-C., Waara I., Fridborg K., Strandberg B., Carlbom U., Jarup L., Lovgren S., Petef M. Crystal structure of human carbonic anhydrase C. Nat. N. Biol. 1972;235:131-137

67. Lindskog S. Structure and mechanism of carbonic anhydrase. Pharmacol. Ther. 1997;74:1-20[Medline]

68. Lippa E. A. The eye: topical carbonic anhydrase inhibitors. Dodgson S. J. Tashian R. E. Gros G. Carter N. D. eds. The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics 1991:171-182 Plenum Press New York.

69. Lovejoy B., Cleasby A., Hassell A. M., Longley K., Luther M. A., Weigl D., McGeehan G., McElroy A. B., Drewry D., Lambert M. H., Jordan S. R. Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. Science (Washington DC) 1994;263:375-377[Abstract/Free Full Text]

70. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature (Lond.) 1991;352:497-505[Medline]

71. Matthews B. W., Schoenborn B. P., Dupourque D., Jansonius J. N., Colman P. M. 3-Dimensional structure of thermolysin. Nat. N. Biol. 1972;238:37-41

72. Matthews R. G., Goulding C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1997;1:332-339[Medline]

73. McCance R. A., Widdowson E. M. The absorption and excretion of zinc. Biochem. J. 1942;36:692-696

74. McGrath M. E., Haymore B. L., Summers N. L., Craik C. S., Fletterick R. J. Structure of an engineered metal-actuated switch in trypsin. Biochemistry 1993;32:1914-1919[Medline]

75. Myers L. C., Terranova M. P., Ferentz A. E., Wagner G., Verdine G. L. Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile. Science (Washington DC) 1993a;261:1164-1167[Abstract/Free Full Text]

76. Myers L. C., Terranova M. P., Nash H. M., Markus M. A., Verdine G. L. Zinc binding by the methylation signaling domain of Escherichia coli Ada protein. Biochemistry 1992;31:4541-4547[Medline]

77. Myers L. C., Verdine G. L., Wagner G. Solution structure of the DNA methyl phosphotriester repair domain of Escherichia coli Ada. Biochemistry 1993b;32:14089-14094[Medline]

78. Park H.-W., Boduluri S. R., Moomaw J. F., Casey P. J., Beese L. S. Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution. Science (Washington, DC) 1997;275:1800-1804[Abstract/Free Full Text]

79. Pauptit R. A., Karlsson R., Picot D., Jenkins J. A., Niklaus-Reimer A.-S., Jansonius J. N. Crystal structure of neutral protease from Bacillus cereus refined at 3.0 Å resolution and comparison with the homologous but more thermostable enzyme thermolysin. J. Mol. Biol. 1988;<199/VOLUME-NR>:525-537[Medline]

80. Pavletich N. P., Pabo C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 Å. Science (Washington, DC) 1991;252:809-817[Abstract/Free Full Text]

81. Peariso K., Goulding C. W., Huang S., Matthews R. G., Penner-Hahn J. E. Characterization of the zinc binding site in methionine synthase enzymes of Escherichia coli: the role of zinc in the methylation of homocysteine. J. Am. Chem. Soc. 1998;120:8410-8416

82. Pearson R. G. J. Am. Chem. Soc. 1963;85:3533-3539

83. Pocker Y., Ng J. S. Plant carbonic anhydrase: hydrase activity and its reversible inhibition. Biochemistry 1974;13:5116[Medline]

84. Rees D. C., Lewis M., Lipscomb W. N. Refined crystal structure of carboxypeptidase a at 1.54 A resolution. J. Mol. Biol. 1983;168:367-387[Medline]

85. Regan L. Protein design: novel metal-binding sites. Trends Biochem. Sci. 1995;20:280-285[Medline]

86. Rowlett R. S. The reversible inhibition of carbonic anhydrase II: computer simulations of a proposed mechanism of action. J. Prot. Chem. 1984;3:369-393

87. Salowe S. P., Marcy A. I., Cuca G. C., Smith C. K., Kopka I. E., Hagmann W. K., Hermes J. D. Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme. Biochemistry 1992;31:4535-4540[Medline]

88. Silverman D. N., Lindskog S. The catalytic mechanism of carbonic anhydrase: implications of a rate-limiting protolysis of water. Acc. Chem. Res. 1988;21:30-36

89. Silverman D. N., Vincent S. H. Proton transfer in the catalytic mechanism of carbonic anhydrase. CRC Crit. Rev. Biochem. 1984;14:207-255

90. Sly W. S., Hewett-Emmett D., Whyte M. P., Yu Y.-S. L., Tashian R. E. Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc. Natl. Acad. Sci. U.S.A. 1983;80:2752-2756[Abstract/Free Full Text]

91. Sly W. S., Hu P. Y. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 1995;64:375-401[Medline]

92. Spurlino J. C., Smallwood A. M., Carlton D. D., Banks T. M., Vavra K. J., Johnson J. S., Cook E. R., Falvo J., Wahl R. C., Pulvino T. A., Wendoloski J. J., Smith D. L. 1.56 Å structure of mature truncated human fibroblast collagenase. Prot. Struct. Funct. Genet. 1994;19:98-109

93. Sutton B. J., Artymiuk P. J., Cordero-Borboa A. E., Little C., Phillips D. C., Waley S. G. An X-ray-crystallographic study of ß-lactamase II from Bacillus cereus at 0.35 nm resolution. Biochem. J. 1987;248:181-188[Medline]

94. Thompson R. B., Ge Z., Patchan M. W., Fierke C. A., McCall K. A., Elbaum D., Christianson D. W. Determination of multiple analytes using a fiber optic biosensor based on fluorescence energy transfer. SPIE 1996;2680:47-56

95. Thompson R. B., Jones E. R. Enzyme-based fiber optic zinc biosensor. Anal. Chem. 1993;65:730-734

96. Vallee B. L. Bertini I. Gary H. B. eds. A synopsis of zinc biology and pathology in zinc enzymes 1986 Birkhauser Boston.

97. Vallee B. L., Auld D. S. Active zinc binding sites of zinc metalloenzymes. Matrix Suppl 1992a;1:5-19[Medline]

98. Vallee B. L., Auld D. S. Active-site zinc ligands and activated H₂O of zinc enzymes. Proc. Natl. Acad. Sci. U.S.A. 1990a;87:220-224[Abstract/Free Full Text]

99. Vallee B. L., Auld D. S. Cocatalytic zinc motifs in enzyme catalysis. Proc. Natl. Acad. Sci. U.S.A. 1993a;90:2715-2718[Abstract/Free Full Text]

100. Vallee B. L., Auld D. S. Functional zinc-binding motifs in enzymes and DNA-binding proteins. Faraday Discuss. Chem. Soc. 1992b;