Zinc-containing Enzyme Research Articles

Crystallization and preliminary X-ray diffraction studies of a novel alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix.

A novel alcohol dehydrogenase enzyme has been cloned from the hyperthermophilic archaeon Aeropyrum pernix and overexpressed in Escherichia coli. This zinc-containing enzyme has been crystallized by the sitting-drop vapour-diffusion method using PEG 600 as precipitant. The crystals diffract to 1.5 A resolution and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 100.7, b = 103.2, c = 67.5 A. The asymmetric unit contains two enzyme monomers. Two synchrotron data sets have been collected: one at a wavelength near the absorption edge of zinc and one at a remote wavelength. Three strong zinc-ion positions were visible in the anomalous Patterson map. Two additional weaker zinc ions have been identified by anomalous Fourier synthesis.

An Observation of a Series of S→N→N Acyl Transfers in 3-Mercaptoproline

Since small peptides do not tend to exist as a single conformation in solution and possess a large amount of flexibility, determination of the preferred peptide structure should be approached by chemical modifications designed to decrease conformational flexibility. The flexibility of a peptide can be limited by utilization of local constraints and cyclization of peptide strand. Also, the incorporation of conformationally constrained amino acids into peptides has been extensively used in the design of conformationally restricted peptides. In order to retain biological activity of parent peptides, the constrained analogs should accommodate the peptide backbone conformation and frequently sustain the crucial functionalities. 1 As a part of our program for targeting zinc-containing enzyme, we designed a series of cysteine-incorporated small peptidomimetic analogs. It is well known that cysteine is the most frequent residue in the catalytic zinc sites of metalloenzymes. 2 In general, zinc ion is likely to coordinate with the cysteine sulfur on the protein/peptide substrate and participate in catalytic and structural sites. 3 This strongly suggested that the cysteine thiol group is one of key elements for enzyme activity. The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. Particularly, we were interested in prolines as conformationally constrained cysteine residues. During the synthesis of 3-mercaptoprolines, we found a serious of acyl transfers. Here, we wish to report the observation of a series of S→N→N acyl transfer in the deprotection of the Sacetate in 3-mercaptoproline systems.

Increased renal vascular resistance in zinc-deficient rats: role of nitric oxide and superoxide.

1. Zinc deficiency (ZD) induces many kinds of pathological states. However, the effects of ZD on haemodynamics remain unclear. In the present study, we measured mean blood pressure (BP) and renal blood flow (RBF) under anaesthesia and calculated renal vascular resistance (RVR) from these parameters in rats maintained on a ZD diet (0.5 p.p.m. zinc) for 4 weeks. 2. Zinc deficiency did not change mean BP, but significantly reduced RBF and increased RVR (each P < 0.01). In addition, these effects of ZD were reversible. 3. Because Cu/Zn superoxide dismutase (SOD) is a zinc-containing enzyme and superoxide is a potent scavenger of nitric oxide (NO), a vasodilator, we hypothesized that one of the mechanisms by which ZD increases RVR is by decreasing NO bioavailability by the enhanced formation of superoxide due to low Cu/Zn SOD activity. To test this hypothesis, we observed the roles of NO and superoxide in the mechanism, after having confirmed the low activity of Cu/Zn SOD in the kidneys of ZD rats. 4. Administration of the SOD mimetic tempol (5 mg/kg per min) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that superoxide was responsible for the mechanism. Low doses of the NO donor sodium nitroprusside (SNP; 2.0 micro g/kg per min, continuous) decreased RVR to a significantly smaller extent in ZD rats compared with control, whereas a high dose of SNP (0.75 mg/kg, bolus) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that the mechanism includes an inhibition of NO activity in ZD, which is most likely to be a scavenging of NO by the activated superoxide. 5. In summary, ZD may increase RVR. The mechanism probably includes changes in NO and superoxide activities.

Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori

Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (α and β), catalyzing the reversible hydration of CO 2. Presumably, the high CO 2 requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO 2 concentration in the investigated range (0.1–10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the α-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the β-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the β-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The α-enzyme was found attached to the surface of the bacterium.

The 1.9 Å Crystal Structure of Heat-labile Shrimp Alkaline Phosphatase

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature.Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9Å. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer.SAP shares the core, an extended β-sheet flanked by α-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of −80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis.

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni2+, Mg2+, Co2+, Mn2+, Ca2+, Sr2+, Cu2+ and Zn2+, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.

Crystallization and preliminary X-ray diffraction analysis of L-aminoacylase from the hyperthermophilic archaeon Thermococcus litoralis.

The enzyme L-aminoacylase catalyses the hydrolysis of N-acyl-L-amino acids from peptides or proteins. The recombinant enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been purified to homogeneity. This zinc-containing enzyme has been crystallized from ammonium sulfate using the sitting-drop vapour-diffusion method. The crystals diffract to 2.8 A resolution and belong to the rhombohedral space group R32, with unit-cell parameters a = b = 102.4, c = 178.5 A, gamma = 120 degrees in a hexagonal lattice setting. The asymmetric unit contains one enzyme monomer, containing a single zinc ion. Two synchrotron data sets have been collected at a remote wavelength and at the maximum f'wavelength for zinc. This has allowed the position of the metal to be identified in anomalous Patterson maps.

Matrix metalloproteinases and matrix metalloproteinase inhibitors in lung cancer

Preclinical studies have provided evidence that matrix metalloproteinases (MMPs), a family of zinc-containing proteolytic enzymes, facilitate tumor invasion, the establishment of metastases, and the promotion of tumor-related angiogenesis. Matrix metalloproteinase inhibitors (MMPIs) have been shown to inhibit tumor growth and dissemination in preclinical models. Not all lung cancers express the MMPs believed to be most important in promoting the neoplastic process, and there are conflicting reports regarding the prognostic significance of MMPs in lung cancer. However, it is possible that these observations are because of limitations in the procedures for measuring MMPs. Many investigators believe that MMPs are universally involved in tumor progression; this hypothesis was the basis for initiating seven phase III MMPI trials in lung cancer. Four studies were closed at completion of the predefined accrual goal, and three were closed early. There were no significant differences in survival in a non–small cell lung cancer prinomastat study, and in a small cell lung cancer marimastat trial. The results of the remaining five studies have not been reported. At this point it appears that MMPIs will probably not play a major role in the treatment of advanced lung cancer patients. Semin Oncol 29 (suppl 4):78-86. Copyright 2002, Elsevier Science (USA). All rights reserved.

Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 A resolution.

Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer. We have determined the first structure of a T-CDA from Bacillus subtilis. The active form of T-CDA is assembled of four identical subunits with one active site apiece. The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site. The similarity results in a conserved structural core in the two CDA forms. An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form. The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion. How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma. However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.

Cloning and characterization of a novel human alcohol dehydrogenase gene (ADHFe1).

There are three types of NAD(P)(+)-dependent alcohol dehydrogenase: "medium-chain" zinc-containing enzymes, "short-chain" zinc-lacking enzymes and iron-activated enzymes. Although the medium-chain family and the short-chain family have been characterized in human, the iron-activated alcohol dehydrogenase family seems to exist only in microbial organisms. We have now cloned and characterized an iron-activated alcohol dehydrogenase gene, Fe-containing alcohol dehydrogenase 1 (ADHFe1) in human. The cDNA was isolated from the human fetal brain cDNA library. It contains a long open reading frame, which is not homologous with the other alcohol dehydrogenases isoenzymes characterized in human. The hypothetical alcohol dehydrogenase does, however, show strong homology to the iron-activated alcohol dehydrogenases from microbial organisms. Northern blotting analysis only detected expression in adult liver tissue. At least two different splicing variants were screened by PCR using the multiple tissue cDNA panels as templates.

Cellular zinc content is a major determinant of iron chelator–induced apoptosis of thymocytes

AbstractDesferrioxamine (DFO) and the hydroxypiridinone (HPO) deferiprone (CP20) chelate iron as well as other metals. These chelators are used clinically to treat iron overload, but they induce apoptosis in thymocytes. Thymocyte apoptosis is potentiated by zinc deficiency, suggesting that these iron chelators may induce apoptosis by depleting stores of zinc. Exposure of murine thymocytes to either DFO or deferiprone resulted in significant reductions in the labile intracellular zinc pool. Moreover, increasing intracellular zinc levels, by chronic zinc dietary supplementation to mice or in vitro loading with zinc, abrogated deferiprone-induced murine thymocyte apoptosis. Bidentate hydroxypyridinones such as deferiprone interact with intracellular zinc pools in a manner distinct from that of DFO, which is a hexadentate iron chelator. Whereas deferiprone acts synergistically with the zinc chelator NNNN-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to induce apoptosis, DFO does not. This difference is most likely due to the ability of HPOs but not DFO to “shuttle” zinc onto acceptors such as metallothioneins. By nature of its structure, DFO is larger than deferiprone and is thus less able to access some intracellular zinc pools. Additionally, metal complexes of DFO are more stable than those of HPOs and thus are less likely to donate zinc to other acceptors. The ability of deferiprone to preferentially access zinc pools was also demonstrated by inhibition of a zinc-containing enzyme phospholipase C, particularly when combined with TPEN. These findings suggest that bidentate iron chelators access intracellular zinc pools not available to DFO and that zinc chelation is a mechanism of apoptotic induction by such chelators in thymocytes.

Syntheses and Kinetic Evaluation of Racemic and Optically Active 2-Benzyl-2-methyl-3,4-epoxybutanoic Acids as Irreversible Inactivators for Carboxypeptidase A

Racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids were synthesized and evaluated as inactivators for carboxypeptidase A, a representative zinc-containing proteolytic enzyme. Only the threo-form of the inactivator is effective and its potency in terms of kinact/KI value is lower by 42-fold compared with 2-benzyl-3,4-epoxybutanoic acid, indicating that the α-methyl group affects adversely in the inactivation contrary to the expectation that it would enhance the inactivation activity of the inhibitor through additional interactions of the methyl group with a small cavity (α-methyl hole) present next to the S1′ hydrophobic pocket. Of the enantiomeric pair, the inactivator having the (2S,3R)-configuration is more potent than its enantiomer by 44-fold. The observed kinetic results may be rationalized on the basis that the methyl group in the inactivator having the (2R,3S)-configuration experiences the van der Waals repulsive interactions with the bottom of the active site crevice in binding to CPA, casting a doubt on the presence of the so-called α-methyl hole at the active site of carboxypeptidase A.

Mechanistic insight into the inactivation of carboxypeptidase A by alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity.

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k(obs)/[I](o)) fall in the range of 1.7-3.6 M(-1) min(-1). The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, alpha-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.

Effects of lead and/or zinc exposure during the second stage of rapid postnatal brain growth on delta-aminolevulinate dehydratase and negative geotaxis of suckling rats.

Lead has been shown to produce cognitive and motor deficits in young rats that could be mediated, at least in part, by inhibition of the zinc-containing heme biosynthetic enzyme delta-aminolevulinate dehydratase (ALA-D). In the present study we investigated the effects of lead and/or zinc treatment during the second stage of rapid postnatal brain development on brain, kidney and blood ALA-D specific activity, as well as the negative geotaxis behavior of rats. Eight-day-old Wistar rats were injected intraperitoneally with saline, lead acetate (8 mg/kg) and/or zinc chloride (2 mg/kg) daily for five consecutive days. Twenty-four hours after treatment, ALA-D activity was determined in the absence and presence of DL-dithiothreitol (DTT). The negative geotaxis behavior was assessed in 9- to 13-day-old rats. Treatment with lead and/or zinc did not affect body, brain or kidney weights or brain- or kidney-to-body weight ratios of the animals. In spite of the absence of effect of any treatment on ALA-D specific activity in brain, kidney and blood, the reactivation index with DTT was higher in the groups treated with lead or lead + zinc than in the control group, in brain, kidney and blood (mean +/- SEM; brain: 33.33 +/- 4.34, 38.90 +/- 8.24, 13.67 +/- 3.41; kidney: 33.50 +/- 2.97, 37.60 +/- 2.67, 15.80 +/- 2.66; blood: 63.95 +/- 3.73, 56.43 +/- 5.93, 31.07 +/- 4.61, respectively, N = 9-11). The negative geotaxis response behavior was not affected by lead and/or zinc treatment. The results indicate that lead and/or zinc treatment during the second stage of rapid postnatal brain growth affected ALA-D, but zinc was not sufficient to protect the enzyme from the effects of lead in brain, kidney and blood.

Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.

The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate, thus affording a direct link between chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.

The design, structure, and therapeutic application of matrix metalloproteinase inhibitors.

Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes involved in the degradation and remodeling of extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMP activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. Thus, MMP inhibitors are expected to be useful for the treatment of these disorders. This article reviews briefly the biochemistry of MMPs and evidence for their pathogenic roles using molecular biology approaches. Biomolecular structures used in the design of MMP inhibitors are thoroughly covered. Major emphasis is on recently published potent, small molecular weight MMP inhibitors and their pharmacological properties. Finally, available clinical results of compounds in development are summarized.

Inhibitors of leukotriene A4 (LTA4) hydrolase as potential anti-inflammatory agents.

Leukotriene A4 (LTA4) hydrolase is a zinc-containing enzyme which stereospecifically catalyzes the hydrolysis of the epoxide LTA4 to the diol leukotriene B4 (LTB4). There is substantial evidence that LTB4 plays a significant role in the amplification of many inflammatory disease states. Therapeutic agents which selectively inhibit LTA4 hydrolase would block the formation of LTB4 and thus be potentially useful for the treatment of inflammation. Numerous inhibitors of LTA4 hydrolase have been reported over the past 15-20 years. Several early inhibitors were based on the structure of the natural substrate, LTA4. Later approaches utilized known inhibitors of related zinc-containing metalloproteinases and led to the identification of captopril, bestatin and kelatorphan as potent inhibitors of LTA4 hydrolase. This led to the design of a number of peptide and non-peptide analogs which contained potential zinc-chelating moieties, including thiols, hydroxamates and norstatines. A more recent series of non-peptidic, non-zinc chelating inhibitors of LTA4 hydrolase has been reported. This work led to the identification of several novel classes of analogs, including imidazopyridines, amidines and cyclic and acyclic amino acid derivatives, ultimately resulting in the identification of two potential clinical candidates SC-56938 and SC-57461A.

Methylation of (2-methylethanethiol-bis-3,5-dimethylpyrazolyl)methane zinc complexes and coordination of the resulting thioether: relevance to zinc-containing alkyl transfer enzymes.

A series of zinc complexes using a new tripodal, N(2)S, heteroscorpionate ligand (L3SH) that is isostructural and isoelectronic with the well-known N(3) trispyrazolylborates have been methylated in solution and the coordination properties of the resulting thioether examined. This system models the reactivity of zinc-containing enzymes involved in alkyl group transfers such as the DNA repair protein Ada from E. coli, or farnasyl transferase where it has been shown that the thioether resulting from alkyl group transfer remains in the coordination sphere of the zinc. The following complexes have been structurally characterized: [(L3S)ZnI] (1), [(L3SCH(3))ZnI(2)] (2), [(L3SCH(3))ZnI]BF(4) (3), [(L3SCH(3))Zn-mu-bis-acetato-mu-hydroxo-Zn(L3SCH(3))]BF(4) (5), [(L3SCH(3))ZnSPh(F5)]ClO(4) (7), and [(L3SCH(3))(2)Zn](BF(4))(2) (8). Complexes 3, 4, 5, 7, and 8 all display thioether coordination. Thus in the absence of superior anionic ligands, thioethers are reasonably good donors to zinc in either a tetrahedral or octahedral geometry. The methylation of the complex [(L3S)ZnSPh(F5)], which contains two different thiols, produces a single product, 7, where only the aliphatic thiol has been alkylated. This observation validates the suggestion that reactivity in enzymes with multiple zinc-bound thiols could be controlled by differences in thiol pK(a) (Hammes, B. S.; Warthen, C. R.; Crans, D.; Carrano, C. J. J. Biol. Inorg. Chem. 2000, 6, 82. Compound 7 is also of interest in that it resembles the metal ion-binding site of the blue copper protein, azurin.

16] Alcohol dehydrogenases from Thermococcus litoralis and Thermococcus strain ES-1

Publisher Summary Alcohol dehydrogenases catalyze the reversible interconversion of alcohols and aldehydes using NAD(P) as the electron carrier. Such enzymes are present in virtually all life forms and can be divided into three different groups based on their molecular properties, Group l contains the long-chain alcohol dehydrogenases represented by horse liver alcohol dehydrogenase, which is a zinc-containing enzyme, Group II contains short-chain alcohol dehydrogenases, such as the one from Drosophila melanogaster, and these lack metals. Group III contains a small number of iron-dependent alcohol dehydrogenases, which are represented by the second alcohol dehydrogenase, ADH2, purified from Zymomonas mobilis. Several alcohol dehydrogenases have been characterized from hyperthermophilic archaea. A zinc-containing group I enzyme was purified from Sulfolobus solfataricus , and a novel type of iron-containing alcohol dehydrogenase has been purified from Thermococcus litoralis and Thermococcus strain ES-1. This chapter describes the methods used to assay, purify, and characterize the iron-containing alcohol dehydrogenases from T. litoralis and Thermococcus strain ES-1.

Methylation of neutral pseudotetrahedral zinc thiolate complexes: model reactions for alkyl group transfer to sulfur by zinc-containing enzymes.

Eight scorpionate-zinc thiolate complexes, [(L1O)ZnSPh], [(L1O)ZnSPhF5], [(L1O)ZnSBz], [(L1O)ZnSPh2,6-Me], [(L1O)ZnSPh2,4-Me], [(L1O)ZnSPh4-NO2], [(TpPh,Ph)ZnSPh], and [(L2S)ZnSPh], were reacted with methyl iodide in chloroform, liberating the corresponding methyl thioethers as determined by 1H NMR. Three of these complexes are new and their synthesis and structural characterization are reported here. Weak alkylating agents such as trimethyl phosphate failed to undergo methyl transfer to the zinc thiolates under these conditions. Analysis of kinetic data as a function of concentration, temperature, pKa of the exogenous thiolate, and donor atom of the tripodal ligand are consistent with a mechanism where the zinc-bound thiolate is the active nucleophile in an associative-type methyl transfer reaction. Our model studies also provide experimental evidence to support the hypothesis that some enzymes can use the charge of the metal coordination site to modulate catalytic activity.