Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency

Kiefer, Laura L.; Paterno, Steven A.; Fierke, Carol A.

doi:10.1021/ja00131a004

Cited by 172 publications

(209 citation statements)

References 9 publications

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“…For example, in the metalloenzyme carbonic anhydrase II, His 94 , His 96 , His 119 , and a water molecule act as primary ligands for the active site zinc ion. However, the affinity of zinc(II) binding can be fine-tuned by manipulating a set of second shell residues, Gln 92 , Asn 244 , Glu 117 , and Thr 199 (53). These residues do not interact directly with the zinc ion but form a hydrogenbond network with the primary shell residues.…”

Section: Discussionmentioning

confidence: 99%

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

Jayasinghe

Miles

Bayley

2006

Journal of Biological Chemistry

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Staphylococcal ␣-hemolysin (␣HL) is a ␤ barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, ␣HL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of ␣HL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of ␣HL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser 217 3 Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of ␣HL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of ␣HL monomers in solution.

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Section: Discussionmentioning

confidence: 99%

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

Jayasinghe

Miles

Bayley

2006

Journal of Biological Chemistry

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“…Substituting residue Glu117 with Ala, Gln, or Asp reduces the rate of association with Zn II by a factor of 10 2 -10 4 . 252,269 Changes in direct ligands have a greater effect on catalytic activity than do changes in indirect ligands: in contrast to mutations of direct ligands (which often practically abolish catalytic activity), the catalytic activity of indirect mutants persists with only a modest 10-fold loss in k cat /K m relative to the wild type (Table 7). An exception is the Glu117Gln mutation, which increases the pK a of Zn II -OH 2 + and abolishes catalytic activity.…”

Section: Effects Of Ligands Directlymentioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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“…Additionally, the exploration of 1,8-di-substituted-10-anthracene sulfonamide-based fluorophores, as suggested by our modeling studies, may yield a probe molecule with fluorescence wavelength properties optimized for use in fiber optic arrays. Future studies will focus on the structure-assisted redesign of both the fluorophore and CAII Ippolito and Christianson, 1994;Kiefer and Fierke, 1994;Ippolito et al, 1995;Kiefer et al, 1995;Lesburg and Christianson, 1995;Nair et al, 1995) in the optimization of a CAII-based zinc biosensor.…”

Section: Figmentioning

confidence: 99%

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

Nair¹,

Elbaum²,

Christianson³

1996

Journal of Biological Chemistry

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The three-dimensional structure of human carbonic anhydrase II (CAII) complexed with the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-Å resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAII, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val-121, Phe-131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Sensors based upon biological macromolecules are increasingly used for the selective detection of numerous chemical entities, including the detection of trace quantities of metal ions. The advantages of metalloprotein-based biosensors over classical fluorometric chemical indicators for such applications include greater recognition and affinity for the target metal ion, exquisite discrimination among transition metals, and kinetically rapid biosensor-analyte association and dissociation. Recently, Thompson and Jones (1993) have exploited the selective recognition of zinc by the metalloenzyme carbonic anhydrase II (CAII) 1 in the design of an enzyme-based zinc biosensor. The physical basis of this sensor is the binding of zinc to the CAII apoenzyme, followed by binding of the fluorophore inhibitor 5-dimethylamino-1-naphthalene sulfonamide (dansylamide, K d ϭ 0.93 M (Nair et al., 1995); see Fig. 1) to the zinccontaining CAII holoenzyme. This leads to a titratable change in the fluorescence emission wavelength and intensity (Chen and Kernohan, 1967); nanomolar concentrations of zinc can be detected and quantified by ratiometric methods by measuring the emission of free dansylamide at 580 nm and the emission of bound dansylamide at 470 nm when excited at 290 nm.The zinc ion of CAII resides at the base of a 15-Å deep cleft, where it is liganded by the imidazole side chains of His-94, His-96, and His-119 (Håkansson et al., 1992). Hydroxide ion, which is the catalytic nucleophile in the CO 2 hydration mechanism, completes a tetrahedral metal coordination polyhedron. Zinc-bound hydroxide donates a hydrogen bond to the hydroxyl group of Thr-199, which in turn donates a hydrogen bond to Glu-106 (Eriksson et al., 1986, 1988aHåkansson et al., 1992). The binding of sulfonamide inhibitors displaces zinc-bound hydroxide and maintains the tetrahedral metal coordination polyhedron by the coordination of an ionized sulfonamide NH group, which also maintains the hydrogen bond interaction with Thr-199 (e.g. see Eriksson et al. (1988b) and Vidgren et al.

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Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency

Cited by 172 publications

References 9 publications

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

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