The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.     

Zinc ion-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime: bimolecular participation of hydroxozinc(II) ion

The Zn²⁺-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime (A) proceeds through three paths. The first two paths, the water path and the hydroxide path, have been observed in related systems. The third path, whose rate is bimolecular with respect to hydroxozinc(II) ion, is newly observed. The methyl group of A raises reaction rates through steric compression. The rate data obtained with A provide further evidences for the intramolecular nucleophilic attack of the metal-bound water molecule or hydroxide ion at the complexed ester. The third path is attributed either to the general base participation of free hydroxozinc(II) ion in the nucleophilic reaction of the Zn(II)-bound hydroxide ion or to the intramolecular reaction of dimeric hydroxozinc(II) ions. Implications of the present results to the actions of metalloenzymes, especially carbonic anhydrase and carboxypeptidase A, are also discussed.     

Metal poison inhibition of carbonic anhydrase

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO₂) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.     

Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Structure of cobalt carbonic anhydrase complexed with bicarbonate.

The three-dimensional structure of a complex between catalytically active cobalt(II) substituted human carbonic anhydrase II and its substrate bicarbonate was determined by X-ray crystallography (1.9 A). One water molecule and two bicarbonate oxygen atoms are found at distances between 2.3 and 2.5 A from the cobalt ion in addition to the three histidyl ligands contributed by the peptide chain. The tetrahedral geometry around the metal ion in the native enzyme with a single water molecule 2.0 A from the metal is therefore lost. The geometry is difficult to classify but might best be described as distorted octahedral. The structure is suggested to represent a water-bicarbonate exchange state relevant also for native carbonic anhydrase, where the two unprotonized oxygen atoms of the substrate are bound in a carboxylate binding site and the hydroxyl group is free to move closer to the metal thereby replacing the metal-bound water molecule. A reaction mechanism based on crystallographically determined enzyme-ligand complexes is represented.     

Mechanism of cyanamide hydration catalyzed by carbonic anhydrase II suggested by cryogenic X-ray diffraction

The three-dimensional structure of a possible intermediate in the hydration reaction of cyanamide to urea catalyzed by human carbonic anhydrase II (hCAII) has been determined by cryocrystallographic techniques. The crystal structure shows that two different adducts are formed under the experimental conditions and that they have different occupancy in the crystal. The high occupancy form consists of a binary hCAII-cyanamide complex where the substrate has replaced the zinc-bound hydroxide anion present in the native enzyme, maintaining the tetrahedral geometry around the metal ion. The second, low-occupancy form consists of a hCAII-cyanamide-water ternary complex where the catalytic zinc ion, still being bound to cyanamide, is approached by a water molecule in a five-coordinate adduct. While the first form can be considered a nonproductive complex, the second form may represent an intermediate state of the catalyzed reaction where the water molecule is about to perform a nucleophilic attack on the zinc-activated cyanamide substrate. The structural evidence is consistent with the kinetic data previously reported about this recently described hydrolytic reaction catalyzed by hCAII, and indicates that a different mechanism with respect to that generally accepted for the physiologic carbon dioxide hydration reaction may be adopted by the enzyme, depending on the substrate chemical properties.     

Inhibition of bacterial peptide deformylase by biaryl acid analogs

Peptide deformylase is an essential eubacterial metalloenzyme involved in the maturation of proteins by cleaving the N-formyl group from N-blocked methionine polypeptides. Biaryl acid analogs containing tetrazole, acyl sulfonamide, or carboxylate pharmacophores were found to be potent inhibitors of recombinant Escherichia coli peptide deformylase. Two of these compounds, a biphenyl tetrazole, compound 1, and a biphenyl acyl sulfonamide, compound 4, were competitive inhibitors with K(i) values of 1.2 and 6.0 microM, respectively. By analogy to the binding of related compounds to other metalloenzymes such as Bacteroides fragilis metallo-beta-lactamase CcrA and human carbonic anhydrase, a mechanism of inhibition is proposed for these peptide deformylase inhibitors where the acidic moieties form direct ionic interactions with the active site metal cation.     

碳酸酐酶Ⅵ的生理功能与酶缺陷性疾病

碳酸酐酶(carbonic anhydrase,CA)催化可逆的水合反应CO2+H2O?ΗCO3?+H+,参与维持pH值平衡、CO2与离子的转运、细胞凋亡等生理过程。碳酸酐酶VI(CA-VI)作为该类含锌酶中惟一的细胞分泌型碳酸酐酶,在哺乳动物及人的唾液腺、乳腺、泪腺、支气管等腺体中表达,对维持口腔、上消化道和呼吸道的生理功能起重要作用。     

The esterase activity of bovine carbonic anhydrase B above pH 9. Reversible and cooalent inhibition by acetozolamide.

The reversible complex between the metalloenzyme bovine carbonic anhydrase B and the sulfonamide inhibitor acetazolamide can be "frozen" irreversibly by the addition of a covalent bond between the methyl group of the inhibitor and the tau-nitrogen of histidine-64. In both cases the inhibited enzyme is inactive as an esterase toward p-nitrophenyl propionate at physiological pH but retains activity controlled by an ionization in the protein exhibiting a pK-a greater than 10. Similarly, both the covalently and reversibly inhibited enzymes in which the catalytically essential Zn(II) ion has been replaced with Co(II) display the same visible absorption spectrum which is invariant over the pH range from 5 to 12. The evidence therefore indicates that the position of the acetazolamide moiety in the active site is independent of both pH and the presence of the covalent bond to histidine-64. Moreover, when reversibly bound, this inhibitor has been shown to replace the water molecule (or hydroxide ion) known to occupy the fourth coordination position of the metal ion and frequently implicated in the catalytic mechanism of carbonic anhydrases. Thus, the activity exhibited by the inhibited enzymes and consequently the second rise observed in the pH rate profile of the native enzyme above pH 0 cannot reflect the ionization of such a water molecule in contrast to what has been postulated previously (Pocker, Y., and Storm, D. R. (1968) Biochemistry 7, 1202-1214). Displacement of the zinc-bound solvent molecule rather than the alkylation of histidine-64 is suggested, however, as the cause of the inactivation of the alkylated enzyme round neutrality. Taken together, the biphasic pH rate profile of native bovine carbonic anhydrase B as well as the activity retained by the alkylated enzyme above pH 9 are best described by a model in which two groups in the enzyme ionize independently, thereby raising the possibility that the high pH activity is controlled by an ionization outside the active site region of the enzyme. Above pH 9.5 the pK; for the reversible interaction between native carbonic anhydrase and acetazolamide falls off linearly with increasing pH. The slope of --1.56 suggests that, among other factors, more than one ionization is responsible for the descending limb of the pH-i-pH profile.     

Studies of Zn(II) and Co(II) complexes of imidazole and n-methylimidazole with regard to the activity related ionization in carbonic anhydrase.

Mixed aquo-N-methylimidazole complexes of Co(II) have been studied as a function of pH to gain a fuller understanding of the metal-binding site in Co(II)-carbonic anhydrase. The inherent affinity of N-methylimidazole for Co(II) has been calculated along with a species distribution for the stepwise addition of ligand to the metal ion. From these studies, it is apparent that the occurrence of Zn(II) rather than Co(II) in native carbonic anhydrase can be explained by the stronger affinity of Zn(II) for imidazole and the preference of Zn(II) for a tetrahedral geometry as offered by the enzyme. Octahedral Co(II) fails to ionize metal bound water. However, at high pH, Co(II)-N-methylimidazole complexes interact directly with the hydroxide ion, generating species with visible spectra very similar to that of Co(II)-carbonic anhydrase. Tentative structures have been proposed for these species.     

Is cyanate a carbonic anhydrase substracte?

A study was undertaken to investigate whether diverse carbonic anhydrase (CA) isozymes (both native Zn as well as cobalt-substituted) are able to catalyze the hydrolysis of anions such as cyanide, cyanate, and thiocyanate. A controversy exists between the crystallographic and spectroscopic data of CA II-anion adducts. In the former case it has been shown that “metal poisons” such as CN⁻and CNO⁻are not directly coordinated to the active site Zn(II) ion whereas spectroscopic studies indicate otherwise. A theoretical study in the above systems did not resolve this controversy, since it was calculated that all three anions can act as CA substrates. In this paper we prove experimentally that none of them may act as substrates of CA and propose an explanation to the above controversy, discussing the mode of binding of small molecules within the enzyme active site. © 1997 Wiley-Liss, Inc.     

Neutral mixed-ligand complexes of 2,2′-bipyridinedichloroplatinum(II) with dianionic aromatic chelating ligands as potential photosensitizers

The electronic spectra of NCS^? and I^? adducts of cobalt(II) human carbonic anhydrase I are pH dependent at pH values below 7. The pK_a of such equilibrium is dependent on the anion concentration and varies between 4.6 and 6.6. The ¹H NMR spectra show that the three histidine residues are bound to the metal ion over the entire pH range investigated. It is supposed that a Glu residue triggers the change in stereochemistry around the metal ion. It is possible that such a Glu residue is Glu 106 present in the active cavity.     

Metalloenzymes and signal transduction: the protein serine/threonine phosphatases,a novel class of binuclear metal-containing enzymes

The serine/threonine protein phosphatases are important regulatory enzymes involved in signal transduction pathways in eukaryotic organisms. These enzymes include protein phosphatases 1, 2A, and 2B (also known as calcineurin). Recent structural data have indicated that the serine/threonine protein phosphatases are novel metalloenzymes containing a dinuclear metal ion cofactor at the active site. The dinuclear metal site is situated in a unique protein fold, a β-α-β-α-β motif which provides the majority of ligands to the metal ions. A similar fold is also seen in plant purple acid phosphatases, which also contain a dinuclear iron–zinc cofactor. In these enzymes, the two metal ions are bridged by a solvent molecule and a carboxylate group from an aspartic acid residue, juxtaposing the two metal ions to within 3.0–4.0?Å of each other. A similar motif has been identified in a number of other enzymes which exhibit phosphoesterase activity, implicating several of them as metalloenzymes which contain dinuclear metal ion cofactors.     

How many carbonic anhydrase inhibition mechanisms exist?

Six genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) were described to date. Inhibition of CAs has pharmacologic applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents. New classes of CA inhibitors (CAIs) were described in the last decade with enzyme inhibition mechanisms differing considerably from the classical inhibitors of the sulfonamide or anion type. Five different CA inhibition mechanisms are known: (i) the zinc binders coordinate to the catalytically crucial Zn(II) ion from the enzyme active site, with the metal in tetrahedral or trigonal bipyramidal geometries. Sulfonamides and their isosters, most anions, dithiocarbamates and their isosters, carboxylates, and hydroxamates bind in this way; (ii) inhibitors that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, 2-thioxocoumarins, sulfocoumarins); (iii) inhibitors which occlude the entrance to the active site cavity (coumarins and their isosters), this binding site coinciding with that where CA activators bind; (iv) compounds which bind out of the active site cavity (a carboxylic acid derivative was seen to inhibit CA in this manner), and (v) compounds for which the inhibition mechanism is not known, among which the secondary/tertiary sulfonamides as well as imatinib/nilotinib are the most investigated examples. As CAIs are used clinically in many pathologies, with a sulfonamide inhibitor (SLC-0111) in Phase I clinical trials for the management of metastatic solid tumors, this review updates the recent findings in the field which may be useful for a structure-based drug design approach of more selective/potent modulators of the activity of these enzymes.     

Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution. Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.     

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.     

Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes.     

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.